1. By use of rabbit isolated perfused intact ears and isolated perfused segments of central and first generation daughter branch ear arteries, we investigated the actions of charybdotoxin (ChTX), a blocker of calcium-activated K+ channels (KCa channels), and N omega-nitro-L-arginine methyl ester (L-NAME) on pressure-flow and diameter-flow relationships. 2. ChTX (1 nM) induced an upwards shift in the pressure-flow curve in the rabbit intact isolated ear preconstricted with 5-hydroxytryptamine (5-HT; 100 nM) with subsequent administration of L-NAME (100 microM) inducing a further upwards shift. L-NAME itself induced an upwards shift in the pressure-flow curve, but subsequent administration of ChTX was without significant effect. 3. Microangiographic analysis revealed a tendency of ChTX (1 nM) to decrease vessel diameter in the central ear artery (G0) with little effect on the first two generations of daughter branch arteries (G1 and G2) in the intact ear. Subsequent addition of L-NAME (100 microM) did not significantly further decrease vessel diameter in G0, but did decrease vessel diameter in G1 and G2. L-NAME itself showed a tendency to decrease vessel diameter in G0, G1 and G2 vessels with subsequent addition of ChTX being without significant effect. 4. In an isolated G0 preparation which was preconstricted with 5-HT (100 nM), ChTX (1 nM) caused an upwards shift in the pressure-flow curve which was augmented by subsequent addition of L-NAME (100 microM). L-NAME (100 microM) itself caused an upwards shift in the pressure-flow curve but subsequent addition of ChTX (1 nM) had no significant effect. 5. In comparison, in an isolated G1 preparation which was preconstricted with 5-HT (100 nM), ChTX (1 nM) had no significant effect on the pressure-flow curve relative to control, but subsequent addition of L-NAME (100 microM) caused an upwards shift. L-NAME (100 microM) itself induced an upwards shift in the pressure-flow curve with subsequent addition of ChTX (1 nM) being without significant effect. 6. ChTX (10 pM-10 nM) caused a concentration-dependent increase in perfusion pressure in isolated G0 and G1 preparations at fixed flow rates of 2 ml min-1 and 0.5 ml min-1, respectively. These responses were enhanced in the presence of L-NAME (100 microM) in G1 but not G0 preparations. 7. We conclude that at 1 nM, ChTX exhibits differential actions on central and daughter branch arteries of the intact ear of the rabbit, which are also apparent in the corresponding arteries when studied in isolation. The action of 1 nM ChTX in G0 vessels may reflect inhibition of either the release or action of nitric oxide as it was blocked in the presence of L-NAME. At higher concentrations of ChTX, there would appear to be a direct constrictor effect on vascular smooth muscle which is apparent in both G0 and G1 vessels. This observed heterogeneity could reflect different distributions of KCa channels between central and daughter branch arteries at either the endothelial or smooth muscle levels, or both.