We investigated whether selective β₁-adrenoceptor stimulation causes hypertrophic growth on isolated ventricular cardiomyocytes from adult rat. As parameters for the induction of hypertrophic growth, the increases of [¹⁴C]phenylalanine incorporation, protein and RNA mass, and cell size were determined. Isoproterenol (Iso, 10 μM) alone had no growth effect. In the presence of the β₂-adrenoceptor antagonist ICI-118551 (ICI, 10 μM), Iso caused an increase in [¹⁴C]phenylalanine incorporation, protein and RNA mass, cell volume, and cross-sectional area. We showed for phenylalanine incorporation that the growth effect of Iso+ICI could be antagonized by β₁-adrenoceptor blockade with atenolol (10 μM) or metoprolol (10 μM), indicating that it was caused by selective β₁-adrenoceptor stimulation. The growth response to Iso+ICI was accompanied by an increase in ornithine decarboxylase (ODC) activity and expression. Inhibition of ODC by the ODC antagonist difluoromethylornithine (1 mM) attenuated this hypertrophic response, indicating that ODC induction is causally involved. The growth response to Iso+ICI was found to be cAMP independent but was sensitive to genistein (100 μM) or rapamycin (0.1 μM). The reaction was enhanced in the presence of pertussis toxin (10 μM). We conclude that selective β₁-adrenoceptor stimulation causes hypertrophic growth of ventricular cardiomyocytes by a mechanism that is independent of cAMP but dependent on a tyrosine kinase and ODC.