Actions of the novel Ca<sup>2+</sup> antagonist, Ro 40-5967, which displays unusual efficacy against endothelin (ET)-induced contractions, were studied in isolated vascular muscle cells (VMCs) using the fluorescent protein kinase C (PKC) indicator, BODIPY 12α-phorbol ester-13β-acetate (PBA-BODIPY). High-sensitivity (photon-counting) digital-imaging microscopy quantified PKC distribution within VMCs and showed translocation from the cytosol to the cell surface membrane on stimulation with ET. ET (1 nM) stimulated translocation of PBA-BODIPY fluorescence that peaked at 4 min, increasing from 19 ± 2% to 29 ± 2% surface membrane (edge) distribution (n = 44, p < 0.05). Increases in membrane-associated PKC due to translocation began within 2 min and persisted for about 10 min, after which a gradual decline to control levels occurred. Upon exposure to Ro 40-5967 (10 µM), there was an inhibition of fluorescence intensity throughout the cell. Average fluorescence intensity decreased to 84 ± 4% (n = 20, p < 0.05) of that in prestimulus controls. Cell/membrane was also reduced to below unstimulated control levels. Amlodipine failed to decrease PKC fluorescence intensity or translocation to the surface membrane. These data suggest that there is an important direct PKC inhibitory action of Ro 40-5967 that would at least partially explain relaxation of ET-induced contractions.