The TASK subfamily of two-pore-domain potassium channels (K2P) encodes for leak K⁺ currents which contribute to the resting membrane potential of many neurons and regulate their excitability. TASK channels are highly regulated by a wide variety of pharmacological and physiological mediators. Currently, there is evidence in the literature both in favour of (Vega-Saenz de Miera et al. 2001) and against (Kim et al. 2000, Meadows & Randall, 2001) regulation of TASK3 currents by protein kinase C (PKC). We address this issue in this study. Whole-cell electrophysiological recordings were made from tsA-201 cells, transiently transfected with wild type and mutated human (h) TASK3 channels. The phorbol ester PMA (100 nM) had no significant effect on hTASK3 mean current amplitude when applied alone (from 1715 ± 112 pA, n = 28, to 1599 ± 84 pA, n = 15, mean ± standard error, p > 0.05, Student’s t test), but reduced mean current amplitude to 619 ± 98 pA (n = 16, p 0.05). The effect of PMA and ionomycin could be completely blocked by the non selective PKC inhibitor BIM (1 μM) (1503 ± 180 pA, n = 7) and by the ‘classical’ PKC inhibitor Go 6976 (100 nM) (1594 ± 144 pA, n = 8). Selective silencing of PKCα activity with a validated siRNA sequence targeted against hPKCα caused a significant reduction in the mean current through hTASK3 channels (1181 ± 140 pA, n = 9) compared to control cells. This current was unaffected, however, following treatment with PMA and ionomycin (1310 ± 136 pA, n = 10, p > 0.05). In contrast, PMA and ionomycin treatment still inhibited hTASK3 current following transfection with a validated siRNA against PKCε (from 1328 ± 105 pA, n = 6 to 580 ± 128 pA, n = 8, p 0.05). Our data show that hTASK3 channels can be strongly inhibited following activation of the classical PKC, PKCα but that the activation requires an elevation of intracellular calcium. Furthermore, PKCα produces this effect on hTASK3 by directly phosphorylating the channel at position T341 in its C terminus.