The site of action of delta-9-tetrahydrocannabinol (THC) to inhibit the release of prolactin (PRL) and growth hormone (GH) was examined by in vivo and in vitro experiments. In conscious freely moving animals bearing implanted third ventricular (3 V) and external jugular cannulae, THC or the diluent was microinjected into the 3 V and blood samples were removed to determine the effect on plasma PRL and GH. Both the 0.4- and 4-µg dose injected intraventricularly resulted in a suppression of PRL and GH release as indicated by declines in plasma levels within 40–80 min which were highly significant statistically but not dose-related. The higher dose evoked a pulse of GH and/or PRL in most animals which preceded the lowering of hormonal levels. In the in vitro experiments dipersed anterior pituitary cells were incubated with 5 × 10–8 or 5 × 10–9M THC or the diluent for 5 days. Fresh culture medium was added to the cells after 3 days and the cells cultured for an additional 2 days. After this period, the cells were incubated for an additional 2 h in culture medium with or without THC plus a near maximal dose of thyrotropin-releasing hormone and GH-releasing factor (50 and 10 ng/ml, respectively) or the diluent to evaluate the response of PRL and GH release, respectively. Neither dose of THC altered the release or storage of the two hormones during culture or affected the response to the releasing hormones which is suggestive that there is no direct effect of THC on either GH or PRL release. The results of the combined in vivo and in vitro studies indicate that the action of THC to suppress PRL and GH release is mediated within the CNS probably by suppression of aminergic activity and peptide