Multiple Ca2+-channels mediate Ca2+ store refilling and agonist induced Ca2+ influx in smooth muscle. We report here that mibefradil, an inhibitor of T type voltage-activated Ca2+-channels, can also inhibit agonist and store mediated Ca2+-entry in arteriolar myocytes. Arterioles (20-40 um outside diameter) were freshly isolated from retinae from humanely killed rats and loaded with fura 2/AM. Mean cytosolic Ca2+ of the smooth muscle cells was estimated by microfluorimetry using 340/380 nm excitation. Single arteriolar fragments were superfused with Ca2+-free Hanks solution and the rate of Ca2+ influx estimated from the rise in fluorescence ratio following re-introduction of Ca2+. Three Ca2+ influx pathways were studied: (i) That due to store refilling after store depletion with cyclopiazonic acid (10 uM, CPA), (ii) agonist induced Ca2+ influx generated by endothelin-1 (Et-1, 10nM) and, (iii) influx initiated by KCl (75 mM). In 1 mM Ca2+, each agent increased Ca2+ influx rate 8-100 fold in different microvessels compared to control. Mibefradil inhibited, dose dependently, all three types of Ca2+ influx pathway at low concentrations with ID50s of 2.3 nM (CPA), 1.2 nM (Et-1) and 6.0 nM (KCl) and 88-96% block at 30 nM (3-5 microvessels from 3-5 animals for each concentration). Perforated voltage clamped recordings were made from single smooth muscle cells within microvessels. No T-type Ca2+ currents were detected on depolarising voltage steps from a holding potential of -100mV even in the presence of a divalent free solution. The retinal arterioles exhibited L-type Ca2+ currents and these were activated by the non-dihydropyridine agonist, FPL-64176 (1 uM). Ca2+ influx rates exceeded the temporal resolution of the drug delivery system (1 s), so Ca2+ influx was studied by introducing 30 uM instead of 1 mM Ca2+. 30 nM mibefradil only reduced this influx rate by 19+2% (n=3). These results suggest that non-voltage operated Ca2+-channels are at least as sensitive to inhibition by mibefradil as classical T type channels.