Adult calcium tolerant rat ventricular myocytes were maintained under serum-free culture conditions for five days. beta-adrenergic and muscarinic cholinergic receptor expression was assessed by radioligand binding determinations using 125I-iodocyanopindolol (ICYP) and [3H]-quinuclidinyl benzilate (QNB), respectively. The binding data were correlated with myocyte structural integrity and contractile responsiveness to norepinephrine (NE). During the 5 days in primary culture, beta-adrenergic and muscarinic cholinergic receptor binding capacity diminished Bmax = 17.1 to 9.2 fmol/mg protein and Bmax = 169.0 to 26.6 fmol/mg protein, respectively. The affinity of both autonomic receptors was unaltered during the period of observation. The majority of isolated myocytes were viable (65 to 85%) and remained rod-shaped for 5 days as assessed by phase contrast microscopy. Up to 2 days in vitro the rod-shaped myocytes appeared ultrastructurally similar to their in vivo counterparts and displayed intact nuclei and the usual complement of cellular organelles. From day 3, phase contrast as well as transmission electron microscopy revealed a progressive increase in autophagic vacuoles consisting primarily of disrupted mitochondria. The number of myocytes that contracted in response to norepinephrine (NE) decreased from 57.2 to 2.3% by day 5. These data indicate that adult rat cardiac myocytes maintained in serum free culture for 5 days, express beta-adrenergic and muscarinic cholinergic receptors. There is a rapid decline (50%) in muscarinic cholinergic receptor number and contractile response to NE by day 2. However, the decrease in beta-receptor Bmax by day two is insufficient to explain the severe loss of cell responsiveness to NE. This functional loss may be related, at least in part, to the ultrastructural abnormalities that are first evident at day 2 in culture. Thus, short-term myocyte cultures that retain phenotypic and physiologic characteristics of in vivo cardiac myocytes could provide a useful in vitro system for exploring pharmacologic-functional interactions in the myocardium.