Calcineurin regulates the proliferation of many cell types through activating the the nuclear factor of activated T-cells (NFAT). Two main isoforms of the calcineurin catalytic subunit (CnA and CnA) have been identified, although their expression and function is largely unknown in smooth muscle. Western blotting and confocal imaging were performed in freshly isolated and cultured rat aortic myocytes to identify these CnA isoforms and elucidate the effect of platelet-derived growth factor (PDGF-BB) on their cellular distribution and interaction with NFAT isoforms. CnA and CnA displayed a differential cellular distribution, with CnA being evenly distributed between the nucleus and cytosol and CnA restricted to the cytosol. In contrast to rat brain, no particulate/membrane localisation of calcineurin was observed. PDGF-BB caused significant nuclear translocation of CnA and induced smooth muscle cell proliferation, effects abrogated by the calcineurin inhibitor, cyclosporin A, the novel NFAT inhibitors, A-285222 and INCA-6 and the adenylyl cyclase activator, forskolin. PDGF also caused cyclosporin-sensitive translocation of NFATc3, with no apparent effect on either CnA or NFATc1 distribution. Moreover, ~87% of nuclear CnA was found to co-localise with NFATc3, consistent with the finding that CnA bound more avidly than CnA to a glutathione S-transferase-NFATc3 fusion protein. Based on their differential distribution in aortic muscle, our results suggest that CnA and CnA are likely to have different functions. However, CnA 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.