Effects of doxorubicin on intracellular Ca2+ concentrations ([Ca2+]i) were examined using myocytes isolated from guinea-pig heart and loaded with fura 2. Changes in twitch contractions were assessed from the motion of myocytes. Ca2+ transients and contractions were triggered by electrical-field stimulation. Exposure of myocytes to doxorubicin depressed Ca2+ transients and contractions. The time to peak Ca2+ transient was prolonged, and Ca2+ sequestration was delayed. In myocytes treated with doxorubicin, an increase in external CaCl2 concentration from 1.2 to 3.6 mM increased the resting and peak [Ca2+]i and enhanced twitch contraction. In the presence of 3.6 mM CaCl2, isoproterenol failed to enhance Ca2+ transients or contractions of doxorubicin-treated myocytes. Effects of doxorubicin were compared with those of agents known to alter Ca2+ handling by myocytes. Caffeine enhanced the peak and resting [Ca2+]2 and contraction. Verapamil caused a rapid decrease in Ca2+ transients and twitch contractions. The effects of verapamil were reversed by isoproterenol in the presence of 3.6 mM CaCl2. Ryanodine alone and combined with doxorubicin depressed contractions and Ca2+ transients and elevated resting [Ca2+]i. Resting [Ca2+]i was further elevated by an increase in CaCl2 concentration and the addition of isoproterenol. The combination of verapamil and ryanodine inhibited Ca2+ transients and contractions. In the presence of verapamil and ryanodine, an increase in extracellular CaCl2 concentration increased resting and peak [Ca2+]i. Isoproterenol further elevated resting and peak [Ca2+]i and increased twitch contractions. These results indicate that doxorubicin alters cellular Ca2+ handling. The actions of doxorubicin are not mimicked by caffeine, verapamil, ryanodine, or the combination of verapamil and ryanodine. Among these agents, ryanodine produced effects that were closest to those observed with doxorubicin.