An increasing number of messengers have recently been found to coexist with growth hormone (GH)-releasing factor (GRF) in hypothalamic neurons. In view of a possible cosecretion of these substances with GRF into the portal circulation, the effect of synthetic rat hypothalamic GRF(1–43) alone, or together with dopamine (DA), L-dopa, γ-aminobutyric acid (GABA), neurotensin (NT) or galanin (GAL) on GH release was investigated by using dispersed rat anterior pituitary cells in monolayer culture. GRF in concentrations of 10-16–10-7M stimulated GH release from somatotrophs in a dose-related manner. DA (10-5 M), L-dopa (10-8 and 10-5-M) and GABA (10-9 and 10-5M) did not affect basal GH release, whereas DA, but not L-dopa or GABA, significantly suppressed GRF-induced GH secretion. However, the inhibitory effect of DA on GRF-stimulated GH secretion was not observed in the presence of somatostatin (10-6M).NT (10-6M) and GAL (10-6M) did not change basal GH release. GAL, but not NT, inhibited GRF-stimulated GH release, but the addition of NT abolished the inhibitory actions of both GAL and DA. These results indicate that substances, probably coreleased with GRF from the same nerve endings, interact in the regulation of GH secretion at the pituitary l