1. The aims of the present study were to investigate the contribution of intracellular calcium and to evaluate the effect of the antagonists of the intracellular calcium stores, thapsigargin and 8-(Diethylamino)-octyl-3,4,5-trimethoxybenzoate, HC1 TMB-8, on phenylephrine-stimulated contractions of rat anococcygeus smooth muscle, using functional studies. 2. Phenylephrine induced concentration-related contractions in both 2.5 mM Ca2(+)-free EGTA media. 3. In Ca2(+)-free media phenylephrine stimulated successive contractions, and the contractile response was abolished only after approximately 26 stimulations. 4. In Ca2(+)-free media, after incubation with 10 microM TMB-8 for 30 min, phenylephrine induced concentration-response curves that shifted to the right. The EC50 values were not changed, and the maximum contractile response was reduced by 39.2 +/- 7.6% in relation to phenylephrine-stimulated responses in absence of TMB-8. 5. Thapsigargin (1 microM) did not alter phenylephrine-stimulated contractions in Ca2(+)-free media. 6. These results indicate that intracellular Ca2+ plays an important role on phenylephrine-stimulated contractions on rat anococcygeus muscle and that the phenylephrine-sensitive intracellular Ca2+ store is not sensitive to thapsigargin.