The aim of the present study was to characterize the receptor subtype and the second messenger involved in the newly discovered hypertrophic effect of beta-adrenoceptor stimulation in cultures of adult ventricular cardiomyocytes. Cardiomyocytes isolated from adult rats and cultured for 6 days in presence of 20% fetal calf serum (FCS) were used as experimental model. Hypertrophic responsiveness of cardiomyocytes was characterized by rate of protein synthesis, increase in protein mass, and increase in RNA content. The hypertrophic effect of the non-specific beta-adrenoceptor agonist isoprenaline was abolished in presence of a specific beta 2-adrenoceptor antagonist (ICI 118,551), could be mimicked by use of a beta 2-adrenoceptor agonist (procaterol) or direct stimulation of adenylate cyclase (forskolin) or addition of a cell-permeable analogue of cAMP (dibuytyrylcyclo-AMP). In presence of Rp-cAMPS, an inhibitor of protein kinase A, the hypertrophic effect of isoprenaline was abolished. The results indicate that the hypertrophic effect of beta-adrenoceptor stimulation is due to stimulation of beta 2-adrenoceptors and activation of adenylate cyclase and protein kinase A.