To examine the molecular mechanisms by which mechanical stimuli induce cardiac hypertrophy and specific gene expression, we cultured rat neonatal cardiocytes in deformable dishes and imposed an in vitro mechanical load by stretching the adherent cells. Myocyte stretching increased total cell RNA content and mRNA levels of c-fos and skeletal alpha-actin. Nuclear run-off transcription assay revealed that this increase in c-fos mRNA level by stretching at least partially reflects changes in the transcriptional status. The transfected chloramphenicol acetyltransferase gene linked to upstream sequences of the fos gene indicated that sequences containing a serum response element were required for efficient transcription by stretching and that sequences containing a cAMP/calcium response element might not be involved in the c-fos response to myocyte stretching. The accumulation of c-fos mRNA by stretching was suppressed by protein kinase C inhibitors at the transcriptional level and inhibited markedly by down-regulation of protein kinase C. Moreover, myocyte stretching increased inositol phosphate levels, and activation of protein kinase C by phorbol esters stimulated the expression of c-fos and skeletal alpha-actin genes. These findings suggest that mechanical stimuli (myocyte stretching) might directly induce cardiac hypertrophy and specific gene expression possibly via protein kinase C activation.