Large conductance, calcium- and voltage-activated potassium (BK(Ca)) channels are important modulators of pulmonary vascular smooth muscle membrane potential, and phosphorylation of BK(Ca) channels by protein kinases regulates pulmonary arterial smooth muscle function. However, little is known about the effect of phosphorylating specific channel subunits on BK(Ca) channel activity. The present study was done to determine the effect of mutating protein kinase C (PKC) phosphorylation site serine 1076 (S1076) on transfected human BK(Ca) channel alpha-subunits in human embryonic kidney (HEK-293) cells, a heterologous expression system devoid of endogenous BK(Ca) channels. Results showed that mutating S1076 altered the effect of PKC activation on BK(Ca) channels in HEK-293 cells. Specifically, the phospho-deficient mutation BK(Ca)-alpha(S1076A)/beta(1) attenuated the excitatory effect of the PKC activator phorbol myristate acetate (PMA) on BK(Ca) channels, whereas the phospho-mimetic mutation BK(Ca)-alpha(S1076E)/beta(1) increased the excitatory effect of PMA on BK(Ca) channels. In addition, the phospho-null mutation S1076A blocked the activating effect of cGMP-dependent protein kinase G (PKG) on BK(Ca) channels. Collectively, these results suggest that specific putative PKC phosphorylation site(s) on human BK(Ca) channel alpha-subunits influences BK(Ca) channel activity, which may subsequently alter pulmonary vascular smooth muscle function and tone.