The mechanisms of verapamil and tetraethylammonium (TEA) inhibition of voltage-gated K⁺ channels in LNCaP human prostate cancer cells were studied in whole-cell and outside/inside-out patch-clamp configurations. Rapidly activating outward K⁺currents (I_K) exhibited neither C-type, nor rapid (human ether á go-go-related gene–type) inactivation. With 2 mM [Mg²⁺]_o, I_K activation kinetics was independent of holding potential, suggesting the absence of etherá go-go-type K⁺ channels. Extracellular applications of TEA and verapamil (IC₅₀ = 11 μM) rapidly (12 s) inhibited I_K in LNCaP cells. Blocking was also rapidly reversible. Intracellular TEA (1 mM), verapamil (1 mM), and membrane-impermeable N-methyl-verapamil (25 μM) did not influence whole-cell I_K, although both phenylalkylamines inhibited single-channel currents in inside-out patches. Extracellular application of N-methyl-verapamil (25 μM) had no influence on I_K. Our results are compatible with the hypothesis that, in LNCaP cells expressing C-type inactivation-deficient voltage-activated K⁺ channels, phenylalkylamines interact with an intracellular binding site, and probably an additional hydrophobic binding site that does not bind charged phenylalkylamines. The inhibiting effects of verapamil and TEA on I_K were additive, suggesting independent K⁺-channel blocking mechanisms. Indeed, TEA (1 mM) reduced a single-channel conductance (from 7.3 ± 0.5 to 3.2 ± 0.4 pA at a membrane potential of +50 mV, n = 6), whereas verapamil (10 μM) reduced an open-channel probability (from 0.45 ± 0.1 in control to 0.1 ± 0.09 in verapamil-treated cells, n = 9). The inhibiting effects of verapamil and TEA on LNCaP cell proliferation were not multiplicative, suggesting that both share a common antiproliferative mechanism initiated through a K⁺ channel block.