We examined the effect of LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, on voltage-dependent K+ (Kv) channels in smooth muscle cells from freshly isolated rabbit coronary arteries using the whole-cell patch clamp technique. The Kv current amplitude was inhibited by LY294002 in a dose-dependent manner, with a Kd value of 1.48 ± 0.20 μM (n=7). Without alteration of the kinetics of activation, LY294002 accelerated the decay rate of Kv channel inactivation. The rate constants of association and dissociation for LY294002 were 1.83 ± 0.01 μM-1s-1 and 2.59 ± 0.14 s-1, respectively (n=6). Application of LY294002 had no significant impact on the steady-state activation (n=6) or inactivation (n=7) curves. In the presence of LY294002, the recovery time constant from inactivation was increased (n=7, P<0.05), and Kv channel inhibition increased under train pulses (1 or 2 Hz, n=6, P<0.05). This indicates that LY294002-induced Kv channel inhibition is use-dependent. Furthermore, pretreatment with another PI3K inhibitor, wortmannin (10 μM), did not affect the Kv current, and did not change the inhibitory effect of LY294002 (n=8). Based on these results, we suggest that LY294002 directly blocks Kv current irrespective of PI3K inhibition. (P<0.05 was regarded significant, and data analysis was done with student t-test.)