OBJECTIVE GH deficiency, either in children or in adults, is a clinically relevant problem. The diagnosis is based on dynamic tests of GH secretion, which are clear cut on a group basis but highly problematic for individual diagnosis. The controversy surrounding the diagnosis of GH deficiency reflects the absence of a gold standard dynamic test. The synthetic hexapeptide hexarelin and GHRH stimulate GH secretion using different mechanisms. A sequential test has been devised using the administration of GHRH as first stimulus followed 120 minutes later by hexarelin. The two aims of the study were (a) to evaluate the interaction of GHRH and hexarelin, and (b) to devise a sequential test of GH reserve.DESIGN The GH stimuli used were GHRH (1 μg/kg i.v.) as a pituitary stimulus, and hexarelin (1 μg/kg i.v.) as a GH stimulus whose main action is hypothalamic. Each subject was tested twice in order to serve as his own control. Three different studies, each with two duplicate tests, were performed on separate groups of individuals: (a) GHRH followed 120 minutes later by hexarelin and on the second day hexarelin followed 120 minutes later by GHRH; (b) GHRH followed 120 minutes later by GHRH and on the other day hexarelin followed 120 minutes later by hexarelin; (c) GH 0.5 IU i.v. followed 120 minutes later by GHRH and on the other day, the same dose of GH followed 120 minutes later by hexarelin.PATIENTS Eighteen normal volunteers (12 women, 6 men) after giving informed consent.MEASUREMENTS Plasma GH levels were measured by time‐resolved fluoroimmunoassay; each value shown is the mean ± SEM ofn = 6.RESULTS GHRH followed 120 minutes later by hexarelin induced two episodes of GH secretion (expressed as mean GH peak, mU/l). The GHRH‐mediated GH release showed a mean GH peak of 38.2 ± 13.6 mU/l and after hexarelin 120 minutes later of 56.7 ± 18.0 mU/l. The contrary sequence blocked the second stimulus, i.e. the hexarelin‐stimulated GH mean peak was 54.7 ± 18.4, and the GH release 120 minutes later after GHRH was 4.8 ± 1.9 (P < 0.05vsGHRH used as first stimulus).In the two sequential tests using the same stimulus, the second GH peak was reduced. In fact, GHRH induced a GH mean peak of 63.8 ± 21.1 mU/l as first stimulus, greater (P < 0.05) than when GHRH was administered again 120 minutes later (22.0 ± 5.9 mU/l). Similar results were obtained with hexarelin, with a first mean peak of 70.6 ± 10.3 mU/l, and a second one 120 minutes later of 13.4 ± 4.6 mU/l (P < 0.05).The blockade of the second stimulus was not due to the feed‐back action of the GH released by the first stimulus. In fact, the i.v. administration of exogenous GH induced a mean GH peak of 168.0 ± 89.7 and reduced the action of GHRH administered 120 minutes later (26.1 ± 8.1). The previous administration of GH (mean peak 115.5 ± 42.0) did not alter the action of hexarelin injected 120 minutes later, showing a mean peak of 71.9 ± 11.2.The large variability in the stimulatory action of GHRH contrasted vividly with the reproducibility of hexarelin. Furthermore, individually analysed, only one of the 12 subjects tested first with hexarelin, compared to 4 out of 12 tested with GHRH as first stimulus, presented a blunted response (<13 mU/l). After the sequential stimulus there were no false negatives.CONCLUSION The sequential administration of GHRH in normal subjects and of hexarelin 120 minutes later provides separate information regarding pituitary GH reserve, of both secretagogues without mutual interference. There were not false negative results to the combi