The protease, a-thrombin (aTh), affects myocardial cell contractility, a feature common among agents that induce hypertrophy. However, it is not known whether cardiac myocytes possess aTh receptors (aTh-R), or if long term treatment with aTh can enhance growth and gene expression. In the present study primary neonatal rat ventricular myocytes expressed a 3.6-kilobase mRNA species that hybridized with a rat aTh-R-specific probe. After 48 h, aTh induced hypertrophy, sarcomeric organization, and enhanced atrial natriuretic factor (ANF) expression, all of which were blocked by the aTh-selective protease inhibitor, D-Phe-Pro-Argchloromethyl ketone. The aTh-R agonist peptide, SFLLRNPND, was a potent activator of ANF expression, however, the non-agonist, FLLRNPND, was inactive. Transfection experiments showed the enhancement of ANF expression by aTh to be transcriptional. The abilities of aTh to induce myocyte hypertrophy and to augment ANF transcription and peptide production were inhibited by the protein kinase C inhibitor, chelerythrine, and by the tyrosine kinase inhibitor, tyrphostin. Thus, myocardial cell aTh-Rs are stimulated by the specific proteolytic actions of aTh, and pathways involving both protein kinase C and protein tyrosine kinases are required for subsequent hypertrophy and ANF expression. Further, these findings suggest a new role for extracellular proteases as regulators of myocardial cell gene expression and growth