The goal was to test whether isoflurane exerts its depressant effect on the heart by mainly affecting the intracellular Ca²⁺ transient [Ca²⁺]_i. Intact rat ventricular trabeculae, paced at 0.5 Hz and 30°C with extracellular [Ca²⁺] ([Ca²⁺]_o) of 2 mM, were used. The [Ca²⁺]_i was monitored using fura 2 injected into the myoplasm. The sarcoplasmic reticulum (SR) Ca²⁺ content was estimated using rapid cooling with or without caffeine to induce Ca²⁺ release and contracture. A plot of peak twitch force versus peak [Ca²⁺]_i transient with increasing isoflurane concentration declines linearly so that a 56% reduction in the peak [Ca²⁺]_i transient would abolish twitch force. This relationship is intermediate between those obtained with lowering [Ca²⁺]_o, which depresses twitch force through a reduction of the [Ca²⁺]_i transient, and adding 2,3-butanedione monoxime, which reduces the responsiveness of the contractile system to [Ca²⁺]_i. The isoflurane effect is different from that of halothane with respect to both the above relationship and the rapid-cooling response. Isoflurane abolishes the ability of rapid cooling to liberate Ca²⁺ from the SR.