The hypothesis that protein kinase C (PKC) and tyrosine kinases, as well as serine–threonine and tyrosine phosphatases, are involved in prolactin (PRL) signalling in theca cells harvested from porcine follicles was tested. Theca cells were incubated with PRL for 24 h to stimulate progesterone (P₄) production. In addition, treatments included inhibitors of PKC and tyrosine kinases, as well as serine–threonine phosphatase inhibitor and tyrosine phosphatase inhibitor. Prolactin significantly stimulated P₄ production by theca cells and all inhibitors suppressed the PRL-stimulated P₄ production. After incubation with PRL for 2, 5, 10 or 20 min, theca cells were homogenized and cytosolic and membrane fractions were obtained. This was followed by determination of PKC activity in partially purified subcellular fractions by measuring the transfer of ³²P from [γ-³²P] adenosine triphosphatase (ATP) to histone III-S. In unstimulated porcine theca cells the major proportion of PKC activity was present in the cytosol. Incubation of cells with PRL resulted in a rapid, time-dependent increase in the amount of PKC activity in the membrane fraction. Protein kinase C activity in the membrane fraction was maximal after 10 min of cells’ exposure to PRL. Protein kinase C activation was assessed also by measuring the specific association of ³H-phorbol dibutyrate (³H-PDBu) with theca cells after treatment with PRL. Prolactin significantly increased ³H-PDBu-specific binding in theca cells. In contrast to PKC, total inositol phosphate accumulation was not affected by PRL in the current study. In summary, PRL stimulated P₄ production by porcine theca cells derived from large follicles. The results of the study were consistent with the hypothesis that PKC is one of the intracellular mediators of PRL action in porcine theca cells. Protein kinase C activation does not appear to occur through the action of phosphatidylinositol-dependent phospholipase C. Moreover, the involvement of tyrosine kinases, as well as tyrosine and serine–threonine phosphatases, in PRL signalling in the examined cells is suggested.