This study investigated whether melatonin could modulate the phosphorylation of the calcium/cyclic AMP response-element binding-protein (CREB) within primary cell cultures of ovine pars tuberalis (oPT) and pars distalis (oPD). Gel shift assays confirmed the presence of nuclear factors able to alter the electrophoretic mobility of a 32P-labelled CRE oligonucleotide. Two shifted bands were observed probably due to monomer and dimer binding to the CRE. Each band was supershifted by antisera directed against both CREB and the phosphorylated form of CREB (P-CREB), consistent with a specific role of CREB proteins in transcriptional regulation. To study the physiological role of CREB, the nuclear immunoreactivity for P-CREB was followed in primary cultures of oPT given different pharmacological treatments. Cells stimulated with forskolin responded with a robust time- and dose-dependent increase in nuclear phospho-CREB immunoreactivity (P-CREB-ir), confirming that activation of this transcription factor occurred through the cyclic AMP-PKA pathway. Maximal stimulation was achieved within 15 min and persisted for up to 1 h. Treatment with melatonin alone did not alter basal P-CREB-ir levels, yet melatonin inhibited the forskolin-induced increase in P-CREB-ir in a dose-dependent manner (IC50 of between 10(-10) M and 10(-8) M melatonin when tested against 1 microM forskolin). In contrast, in primary cultures of oPD, melatonin failed to block forskolin-stimulated increases in either the content of cyclic AMP or the intensity of nuclear P-CREB-ir, confirming that the action of melatonin upon P-CREB-ir is tissue specific. These results demonstrate that, consistent with its inhibitory effect on the activation of PKA within oPT, melatonin prevents or reverses the phosphorylation of CREB induced by activation of the cyclic AMP signal transduction pathway. Therefore melatonin has the potential to regulate gene expression in the oPT by acting upon the CREB transcription factor. However, this paper also shows that 12-O-tetradecanoylphorbol-13-acetate (TPA) which activates PKC also leads to the phosphorylation of CREB in oPT cells, suggesting the potential involvement of other signal transduction pathways in the transcriptional regulation of these cells.