The effects of neutralized acid extracts of rat hypothalami (HE) on the release of bioactive and immunoactive rat growth hormone (GH) were investigated in 4 in vitro experiments. In 2 short-term incubations and in a 3-day organ culture, HE from hypophysectomized rats stimulated the release of biologically active GH from rat adenohypoph-yses, whereas the radioimmunoassay (RIA) recorded increased GH secretion by HE in only 2 of the experiments, and to a lesser degree. Addition of HE to secreted GH strongly inhibited the response of the bioassay animals to the GH, while it had no effect on the measurement of the hormone in the RIA, suggesting that the discrepancies between the amounts of bio- and immunoactive GH secreted as a result of HE were even greater than our assays recorded. The possibility that the inhibition of the measurement of GH in the bioassay was due to low molecular weight substances was tested in an additional short-term incubation experiment. HE from intact rats caused no stimulation of GH release as measured by either assay. Dialysis caused a loss of RIA-detectable GH by about 35% in both the control and HE-containing media, while it had no effect on bioactive GH in control medium. Dialysis did enhance the biological GH activity of the HE medium, however. Thus, a dialyzable material present in HE was interfering with the response of the bioassay animals to GH. These results support other evidence for the differential release of rat GH with varying immuno- and bioactivities and indicate that HE contains low molecular weight substance(s) that inhibit the tibial growth response in hypophysectomized rats to GH.