Calcineurin regulates the proliferation of many cell types through activation of the nuclear factor of activated T cells (NFAT). Two main isoforms of the calcineurin catalytic subunit [calcineurin A (CnA)alpha and CnAbeta] have been identified, although their expression and function are largely unknown in smooth muscle. Western blot analysis and confocal imaging were performed in freshly isolated and cultured rat aortic myocytes to identify these CnA isoforms and elucidate the effect of PDGF on their cellular distribution and interaction with NFAT isoforms. CnAalpha and CnAbeta isoforms displayed differential cellular distribution, with CnAalpha being evenly distributed between the nucleus and cytosol and CnAbeta being restricted to the cytosol. In contrast with the rat brain, we found no evidence for particulate/membrane localization of calcineurin. PDGF caused significant nuclear translocation of CnAbeta and induced smooth muscle cell proliferation, with both effects being abrogated by the calcineurin inhibitor cyclosporin A, the novel NFAT inhibitors A-285222 and inhibitor of NFAT-calcineurin association-6, and the adenylyl cyclase activator forskolin. PDGF also caused cyclosporin A-sensitive translocation of NFATc3, with no apparent effect on either CnAalpha or NFATc1 distribution. Moreover, approximately 87% of nuclear CnAbeta was found to colocalize with NFATc3, consistent with the finding that CnAbeta bound more avidly than CnAalpha to a glutathione S-transferase-NFATc3 fusion protein. Based on their differential distribution in aortic muscle, our results suggest that CnAalpha and CnAbeta are likely to have different cellular functions. However, CnAbeta appears to be specifically activated by PDGF, and we postulate that calcineurin-dependent nuclear translocation of NFATc3 is involved in smooth muscle proliferation induced by this mitogen.