The effects of beta 2- and beta 1-adrenoceptor (beta 2AR and beta 1AR, respectively) agonists on the cytosolic Ca2+ (Cai) transient (indexed by the transient increase in indo-1 fluorescence ratio after excitation), twitch amplitude (measured via photodiode array), membrane potential, and L-type sarcolemmal Ca2+ current (ICa, measured by whole-cell patch electrode) were assessed in single rat ventricular myocytes. The selective beta 2AR agonist Zinterol increased the amplitudes of both the Cai transient and twitch in a concentration-dependent manner. Similar results were obtained when beta 2ARs were stimulated with isoproterenol in the presence of the selective beta 1AR antagonist CGP 20712A. beta 1AR stimulation induced by norepinephrine increased twitch amplitude to about the same extent as did beta 2AR stimulation. However, several striking differences between response to beta 1AR and beta 2AR stimulation were observed. beta 1AR stimulation had the potent effect of abbreviating the time course of the contraction and Cai transient, and beta 2AR stimulation did not reduce the time course of the Cai transient and had only a minor effect on the twitch duration. For a given increase in twitch amplitude, beta 1AR stimulation caused a greater increase in Cai transient, suggesting a diminished Cai-myofilament interaction. beta 1AR, but not beta 2AR, stimulation evoked spontaneous Cai oscillations, increased the diastolic indo fluorescence level, and caused a decline in resting cell length. beta 1AR and beta 2AR also differed in their effects on ICa. Whereas both beta 1AR and beta 2AR stimulation increased the peak ICa amplitude, beta 2AR stimulation markedly prolonged the ICa inactivation time. Accordingly, beta 2AR stimulation prolonged the action potential duration to a greater extent than did beta 1AR stimulation. 8-(4-Chlorophenylthio)cAMP mimicked the effects of beta 1AR stimulation by norepinephrine but not those due to beta 2AR stimulation. These results clearly indicate that both beta 2ARs and beta 1ARs functionally coexist in rat ventricular myocytes but that stimulation of these receptor subtypes elicits qualitatively different cell responses at the levels of ionic channels, the myofilaments, and sarcoplasmic reticulum.