Smooth muscle cells express a variety of voltage-independent Ca²⁺ channels but studies on the functional role of these has been hampered by their confused pharmacological susceptibilities. Previously, we showed that nifedipine in rabbit choroid arterioles, and mibefradil in rat retina appeared to block store-, receptor- and L-type Ca²⁺ channels at the same equally low concentrations. Here we used a range of classical Ca²⁺ channel blockers to identify agents which might be useful in delineating voltage-dependent and -independent Ca²⁺ channels in the same arteriolar smooth muscle cells. Arteriolar fragments (15-35 μm outside diameter) were isolated by gentle trituration of the retinae removed immediately from humanely killed rats. After loading with fura-2 AM, cytosolic Ca²⁺ was measured in the smooth muscle layer by microfluorimetry. Ca²⁺ influx was measured as the rate of Ca²⁺ rise on adding 1 mM to the Ca²⁺-free medium normally superfusing the microvessels. This was performed in the presence of either: (a) 70 mM KCl (to open voltage-dependent Ca²⁺ channels), (b) cyclopiazonic acid (10 Mμ CPA, to stimulate store-depletion operated Ca²⁺ channels), or (c) endothelin-1 (10nM Et-1, to activate receptor-controlled Ca²⁺ influx). Concentration-effect curves were constructed for the Ca²⁺ channel blockers and these used to read off ID₅₀s (Table 1). The effects of SKF96365 and LOE908 were not consistent with their specific actions on either store or receptor induced Ca²⁺ influxes. In this preparation store-operated channels were resistant to nifedipine in contrast to rabbit choroidal arterioles. The results with diltiazem and verapamil suggest that these cells have at least three voltage-independent Ca²⁺ influx pathways which can be separated pharmacologically. Two of these are store-depletion activated channels and can be separated by their differential sensitivity to diltiazem but both have poor sensitivity to verapamil. The receptor-operated component is highly sensitive to both drugs. TRP channels are thought to underlay voltage-indendent Ca²⁺ influx pathways in smooth muscle and a similar screening approach of known Ca²⁺ channel blockers could be a useful strategy to find pharmacological tools which can be used to associate functions with particular molecular entities.