A pplication of mechanical strain to neonatal rat ventricular myocytes in culture evokes changes in gene expression reminiscent of those that occur with hypertrophy in vivo, such as stimulation of brain natriuretic peptide (BNP) gene expression. Here, we show that a major component of strain-dependent BNP promoter activation results from stimulation of p38 mitogen-activated protein kinase (MAPK) in the cardiac myocyte. Strain increased p38 activity in a time-dependent fashion. The p38 inhibitor SB203580 led to a reduction of approximately 60% in strain-activated human BNP (hBNP) promoter activity. Cotransfection of wild-type p38 increased both basal and strain-dependent promoter activity, while cotransfection with MKK6AL, a dominant-negative inhibitor of p38 MAPK kinase, resulted in partial inhibition of either p38- or strain-activated hBNP promoter activity. p38 MAPK increased hBNP promoter activity through activation of the transcription factor NF-κB. Activation of the hBNP promoter by either p38 or strain was mediated by DNA elements present in the 5′ flanking sequence of the gene. Mechanical strain promoted assembly of NF-κB components on these DNA elements in vitro. Thus, induction of the hBNP promoter by mechanical strain depends, at least in part, on stimulation of p38 and subsequent activation of NF-κB. This activation may play an important role in signaling the increased BNP gene expression that accompanies hemodynamic overload and cardiac hypertrophy in vivo.J. Clin. Invest. 104:1603–1612 (1999).