Rat anterior hemipituitaries were incubated in Krebs-Ringer bicarbonate containing [3H]leucine. Newly synthesized [3H]prolactin and [3H]GH in the pituitary and incubation medium were assayed, as was the radioimmunoassayable prolactin released into the medium during a 5-h incubation. Dopamine (7.5 X 10(-8)M), ergocryptine (4 X 10(-10) M) and apomorphine (6 X 10(-8)M) all significantly inhibited both radioimmunoassayable prolactin release and newly-labeled [3H]prolactin release without affecting [3H]GH release. Conversely, dibutyryl cyclic AMP (2.5 mM) stimulated radioimmunoassayable prolactin release as well as [3H]prolactin and [3H]GH release. The addition of 2.5 mM dibutyryl cyclic AMP to media containing dopamine, ergocryptine or apomorphine completely restored both radioimmunoassayable prolactin release and [3H]prolactin release to at least control levels. Dopamine, ergocryptine and apomorphine all inhibited incorporation of [3H]leucine into prolactin but not into GH, whereas 2.5 mM dibltyryl cyclic AMP with any one of the inhibitors restored total incorporation into [3H]prolactin to levels insignificantly lower than the nucleotide-stimulated incorporation. Adenosine and guanosine at 2.5 mM also stimulated incorporation into [3H]prolactin and blocked the inhibitory effects of apomorphine upon [3H]prolactin synthesis and release. These nucleosides also stimulated [3H]GH release; and guanosine, but not adenosine, stimulated incorporation into [3H]GH. The ability of dibutryl cyclic AMP to block the effects of dopamine, ergocryptine and apomorphine upon prolactin release is consistent with these three inhibitors acting by a common mechanism. Cyclic AMP could be hypothesized as a second messenger for prolactin release, but the ability of adenosine and guanosine to mimic almost perfectly the effects of this cyclic nucleotide does not allow any conclusive interpretation.