The Na(+)-Ca(2+) exchanger is crucial in the regulation of [Ca(2+)](i) in the cardiac myocyte. The exchanger is upregulated in cardiac hypertrophy and failure. This upregulation can have an effect on calcium transients and possibly contribute to diastolic dysfunction and an increased risk of arrhythmias. Here we use adenovirus mediated gene expression to examine the role of p38 MAP kinase in upregulation of the exchanger in adult cardiac myocytes. We demonstrate that p38 mediates a part of the alpha-adrenergic stimulated upregulation of the Na(+)-Ca(2+) exchanger gene. Overexpression of dominant-negative p38 isoforms and activated MKK3 and MKK6 in isolated adult cardiac myocytes demonstrates that p38 activation is sufficient for NCX1 promoter upregulation and that this is mediated primarily by the p38alpha isoform. Lastly, this work demonstrates that the p38alpha stimulated upregulation of the NCX1 promoter is mediated via the -80 CArG box element. This is the first time that a specific role for p38alpha in gene regulation has been demonstrated in isolated adult cardiomyocytes and provides an important clue to our understanding some of the factors regulating exchanger gene expression in the hypertrophic and failing heart.