Despite the interest in the roles that mitogen-activated protein kinases (MAPKs) play in the heart, the role of the different MAPK isoforms has been relatively poorly defined. A third isoform of p38 MAPK, known variously as stress-activated protein kinase-3 (SAPK3), p38- γ or ERK6, has been previously shown to differ from p38-α / β both in its molecular weight and its lack of inhibition by the compound SB203580. We have generated monoclonal antibodies with specificity for SAPK3 demonstrated by immunoblot analysis, immunofluorescence studies, and cloning of SAPK3 from a rat heart cDNA expression library. By immunoblotting, we confirmed high expression of SAPK3 in fast, slow and mixed fibre types of murine skeletal muscle and observed significant expression restricted to heart, lung, thymus and testes. In addition to expression in normal heart (human, mouse, rat, dog and pig), we observed constant expression in diseased human heart, as well as control and hypertrophic cultured neonatal rat cardiac myocytes. Immunolocalization in cultured cardiac myocytes followed by confocal microscopy showed punctate, non-nuclear SAPK3 staining. In contrast, p38- α/ β staining was non-punctate and distributed throughout the cytosol and nucleus. Whereas treatment with Leptomycin B to prevent nuclear export processes promoted higher levels of p38- α/ β staining in cardiac myocyte nuclei, there was no apparent change in SAPK3 localization under these conditions. These differences between p38- α/β and SAPK3 probably reflect the specialized functions of SAPK3 and emphasize the need to evaluate SAPK3 upstream activators and downstream targets in the heart.