To investigate the mechanisms of action of ryanodine and caffeine, changes in mechanical and electrical activity caused by these agents were correlated with alterations in45Ca fluxes and cell Ca contents in chick embryo ventricular cell monolayer cultures. Ryanodine (10−10-10−5M) irreversibly decreased contraction amplitude by 10–70% relative to control in a concentration-dependent manner with minimal effects on electrical activity. Ryanodine caused a slight decrease in rapid45Ca uptake, but no change in total exchangeable calcium content or rapid45Ca efflux. Caffeine (1-20 mM) caused a transient (less than 10 seconds) 5–12% increase in contraction amplitude followed by a sustained 9–76% decrease in contraction amplitude and a 10 mV decrease in diastolic membrane voltage. Caffeine caused a decrease in rapid45Ca uptake, a decrease in total exchangeable calcium content, and an increase in rapid45Ca efflux. These results suggest that caffeine produces a decrease in sarcoplasmic reticulum (SR) Ca2+uptake, and/or an increase in SR Ca2+release that eventually depletes the SR of Ca2+, presumably accounting for the negative inotropic effect. The ryanodine effects on contraction are more difficult to account for solely in terms of alterations of transsarcolemmal Ca2+fluxes and Ca2+contents. Our data indicate an important role for the SR in excitation-contraction coupling in cultured chick embryo ventricular cells and suggest that SR Ca2+is part of the rapidly exchanging Ca2+compartment noted in45Ca flux studies.