Voltage-gated K⁺ (Kv) channels of arterial smooth muscle modulate arterial tone, and are inhibited by vasoconstrictors. In rat mesenteric arterial smooth muscle cells both angiotensin II (Ang II) and endothelin-1 (ET-1) inhibit Kv channels through PKC (Hayabuchi et al., 2001; Rainbow et al., 2006) and here we have investigated the PKC isoenzymes involved. Smooth muscle cells were isolated enzymatically from rat mesenteric arteries and expression of PKCs α, δ, ε and λ/ζ was detected using western blotting. Kv currents were recorded in physiological K⁺ concentrations using conventional whole-cell recording where the pipette solution contained 100 nM Ca²⁺ (buffered with 10 mM EGTA), 1 mM Mg²⁺, 1 mM ATP and 0.5 mM GTP. Ang II (100 nM) and ET-1 (10 nM) inhibited Kv currents by similar amounts (52.6 ± 6.7% and 53.2 ± 7.7%, n = 5, 6). However, PKCε inhibitor peptide, added either by inclusion in the patch pipette or externally in the form of a membrane permeable Tat-linked peptide, reduced inhibition by Ang II to 10.6 ± 2.1% (p<0.001 vs control) and 5.3 ± 2.8% (p<0.001) respectively. However, we saw little effect of PKCε inhibitor peptide (included in the pipette) on the inhibition of Kv currents by ET-1. In contrast, Gö6976, an inhibitor of conventional PKCs did not significantly affect Kv inhibition by angiotensin, but reduced that by ET-1 to 17.0 ± 2.8% (p<0.001). To investigate the possible involvement of PKCβ, we tested the PKCβ inhibitors LY333531 and PKCβ inhibitor peptide, neither of which reduced Kv inhibition by endothelin. We also investigated the roles of PKC isoenzymes in the contractile effects of these vasoconstrictors using myography of small mesenteric arterial segments. PKCε inhibitor peptide (Tat-linked to allow intracellular access) substantially inhibited contractions to angiotensin, but did not affect those to ET-1, while Gö6976 inhibited contractions to ET-1 and also reduced those to Ang II by a lesser extent. Our results confirm that Ang II inhibits Kv channels through PKCε, and suggest that ET-1 acts primarily though a conventional PKC, most likely PKCα. These results are also consistent with Kv inhibition contributing to the contractile actions of these vasoconstrictors.