The effect of halothane on the availability of different sources of activator calcium for myocardial contraction was evaluated in relationship to the negative inotropic effect on the anesthetic. This was studied in rabbit papillary muscle under various stimulation conditions that are known to vary the importance of one or another of two sources of Ca2+ for production of contraction. The rested-state contraction is known to depend primarily on the transsarcolemmal influx of Ca2+ for its activation whereas the potentiated-state contraction is dependent largely on Ca2+ released from intracellular stores. In the presence of 0.6% halothane (gas phase) the rested-state and potentiated-state contractions were depressed (mean +/- SEM) 38% +/- 5% (n = 15) and 32% +/- 3% (n = 7), respectively (no statistically significant difference). Halothane accelerated the decay of the potentiated state, indicating that it stimulates the loss of Ca2+ from internal stores. Halothane also acted to slow the time course of force increase following restimulation of rested muscles, indicating that it inhibits the filling of internal stores. These observations suggest that the negative inotropic effect of halothane is caused by a combination of quantitatively similar effects of the anesthetic on the transsarcolemmal and intracellular sources of activator Ca2+.