The effect of halothane on intracellular Ca2+ concentration ([Ca2+](i)) regulation in porcine tracheal smooth muscle cells was examined with real-time confocal microscopy. Both 1 and 2 minimum alveolar concentration (MAC) halothane increased basal [Ca2+](i) when Ca2+ influx and efflux were blocked, suggesting increased sarcoplasmic reticulum (SR) Ca2+ leak and/or decreased reuptake. In β-escin-permeabilized cells, heparin inhibition of inositol 1,4,5-trisphosphate-receptor channels blunted the halothane-induced increase in [Ca2+](i). Both 1 and 2 MAC halothane decreased the frequency and amplitude of ACh-induced [Ca2+](i) oscillations (which represent SR Ca2+ release through ryanodine-receptor channels), abolishing oscillations in ~20% of tracheal smooth muscle cells at 2 MAC. When Ca2+ influx and efflux were blocked, halothane increased the baseline and decreased the frequency and amplitude of [Ca2+](i) oscillations, inhibiting oscillations in ~70% of cells at 2 MAC. The fall time of [Ca2+](i) oscillations and the rate of fall of the [Ca2+](i) response to caffeine were both increased by halothane. These results suggest that halothane abolishes agonist-induced [Ca2+](i) oscillations by 1) depleting SR Ca2+ via increased Ca2+ leak through inositol 1,4,5-trisphosphate- receptor channels, 2) decreasing Ca2+ release through ryanodine-receptor channels, and 3) inhibiting reuptake.