It has been suggested that L-type Ca²⁺ channels may play an important role in the cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca²⁺ channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca²⁺ channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling, via protein kinase activation. Ba²⁺ currents (I_Ba) through L-type Ca²⁺ channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein, using the conventional whole-cell patch clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca²⁺ channel activity, but did not affect the reversal potential and the steady state inactivation. Bath application of selective inhibitors of protein kinase C (PKC), Ro 31-8425 or Go 6983, prevented I_Ba enhancement by hypotonic swelling, while the specific protein kinase A inhibitor KT 5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased I_Ba under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on IBa when cells were first exposed to hypotonic solution. Furthermore, down-regulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca²⁺ channel activity in rabbit portal vein smooth muscle cells through activation of PKC.