Although agonist stimulation leads to an increase in inositol 1,4,5-trisphosphate (InsP3) and decreased calcium in peripherally and centrally located sarcoplasmic reticulum in smooth muscle, the distribution of InsP3 receptors is unknown. InsP3 receptor and the calcium binding protein, calsequestrin were localized by immunolabelling in a tonic and a phasic smooth muscle. InsP3 receptor labelling was predominantly localized at the cell periphery, where most of the sarcoplasmic reticulum is localized in vas deferens (phasic muscle). Elements of central sarcoplasmic reticulum, where present, were also labelled. Distribution of calsequestrin in vas deferens was similar to that of the InsP3 receptor. In aorta (tonic muscle) the InsP3 receptor labelling was proportional to sarcoplasmic reticulum distribution: predominantly central. No labelling of sections or immunoblots was observed with the anti-calsequestrin antibody in aorta. InsP3 and caffeine, but not cyclic ADP-ribose, released intracellular Ca2+ in permeabilized vas deferens and aorta. The ultrastructure of the sarcoplasmic reticulum, investigated in stereo views of semi-thick and thin sections of osmium ferricyanide stained tissue, is shown to have several distinctive features, such as fenestrated sheets (single or in stacks), as well as numerous regions of continuity between central and peripheral sarcoplasmic reticulum, suggesting a single compartment within the smooth muscle cell. Regions of the sarcoplasmic reticulum were closely apposed to and often ensheathed mitochondria. We conclude that InsP3 receptors are present in both the central and the peripheral sarcoplasmic reticulum of tonic and phasic smooth muscle, consistent with electron probe analysis results showing calcium release from both regions.