The cell length of single, isolated rat and dog heart cells was monitored during exposure to halothane-containing solution to define the cellular mechanism of halothane's negative inotropic effect. Spontaneous contractile waves, which reflect spontaneous Ca release from the sarcoplasmic reticulum (SR) in resting rat heart cells, exhibited a significant increase in frequency and a decrease in amplitude in the presence of halothane 0.27 mM (0.9 vol%) and 0.55 mM (1.7 vol%). Electrically stimulated dog and rat heart cells abruptly exposed to halothane (0.47–0.55 mM or 1.5–1.7 vol%) revealed a transient increase in twitch amplitude (significantly different from control). Twitch amplitude then declined to values significantly below control as halothane exposure continued. This decrease in twitch reached 42 ± 13% (mean ± SD) of control in rat cells and 50 ± 14% in dog cells beating at 60 beats per min. In dog cells the magnitude of the transient increase in twitch amplitude was greater at faster beating rates compared with lower rates in the same cells (P< 0.01) and the transient increase was insensitive to verapamil. Halothane 0.55 mM (1.7 vol%) also significantly accelerated the rate of decline in the twitch amplitude of successive beats in rat cells stimulated after a rest interval (negative staircase). The findings regarding spontaneous contractile waves indicate a direct effect of halothane at the SR in resting cells, occurring independently of any changes in the slow inward current. The halothane-induced changes in beating cells can be explained by an enhancement of Ca release from the SR with an eventual reduction of SR Ca stores.