Retinoic acid receptor α (RARα) is required for normal testis function. Similar to other steroid hormone receptors, RARα appears to undergo an activation process by which it translocates from the cytoplasm to the nucleus where it acts as a transcription factor. In this report, we demonstrate that RARα nuclear trafficking in Sertoli cells is positively regulated by phorbol-12-myristate-13-acetate-activated protein kinase C without the requirement of ligand, retinoic acid. Protein kinase C then stimulates the downstream mitogen-activated protein kinase, and the nuclear localization of RARα is dependent on activation of both kinases. The increase in RARα nuclear translocation is also coupled with enhanced transcriptional activity of RARα. This mechanism of RARα positive regulation is unique, different from that of its negative regulation, that has previously been shown to be dependent on cAMP-dependent protein kinase A and more importantly, dependent on its ligand. However, the mechanism by which retinoic acid positively influences the nuclear localization of RARα is not due to retinoic acid directly increasing protein kinase C or mitogen-activated protein kinase activities. Nonetheless, the positive influence of retinoic acid is also dependent on these two kinases as determined by inhibitor studies. These results suggest two mechanisms for RARα activation in Sertoli cells: one involving only the two kinases, the other involving both the ligand and the two kinases. These regulatory mechanisms for RARα activation, both positive and negative, may be critical for the proper function of RARα in the testis.