The present study examined the intracellular signaling mechanisms for the P-glycoprotein (P-gp)-induced modulation of regulatory volume increase (RVI) in the isolated nonperfused proximal tubule S2 segments from wild-type (WT) mice and those (KO) in which both the mdr1a and mdr1b genes were knocked out. The exposure of the tubules from the KO mice to the hyperosmotic solution (500 mOsm/kgH₂O) involving 200 mM mannitol elicited RVI following the initial cell shrinkage, whereas the exposure of the tubules from the WT mice to the hyperosmotic solution did not. The addition of the two P-gp inhibitors (verapamil and cyclosporin A) to the tubules from the WT mice resulted in RVI, whereas the exposure of the tubules from the KO mice to the hyperosmotic solution in the presence of the two P-gp inhibitors had no effect on RVI. The peritubular addition of the protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate, PMA), but not the inactive phorbol (4-phorbol), to the tubules of the WT mice elicited RVI. When the tubules of the WT mice were exposed to the hyperosmotic solution including PMA plus either of the PKC inhibitors (staurosporine and calphostin C), they did not exhibit RVI. In addition, when the tubules of the WT mice were exposed to the hyperosmotic solution involving either of the P-gp inhibitors plus either of the PKC inhibitors, they did not exhibit RVI. In the tubules of the KO mice, the peritubular addition of the PKC inhibitors abolished RVI, but PMA did not. The exposure to the hyperosmotic mannitol solution activated PKC in the proximal straight tubules from the KO mice, but not from the WT mice. In the tubules of the WT mice, the hyperosmotic mannitol solution including PMA also activated PKC. In the tubules of the WT mice, the peritubular addition of the microtubule disruptor (colchicine), the microfilament disruptor (cytochalasin B), the phosphatidylinositol 3-kinase (PI 3-kinase) blocker (wortmannin), but not another PI 3-kinase blocker (LY 294002), inhibited the PMA-induced RVI. In the tubules of the KO mice, the peritubular addition of colchicine, cytochalsin B, and wortmannin abolished RVI, but LY 294002 did not. We conclude that 1) in the mouse proximal tubule, P-gp-induced modulation of RVI during the exposure to the hyperosmotic mannitol solution is mediated via PKC, and 2) the microtubule, microfilament, and wortmannin-sensitive, LY 294002-insensitive PI 3-kinase contribute to the PKC-induced RVI.