The objective of this study was to examine whether ion pair complexation with endogenous bile salts in hepatocytes contributes to the preferential biliary excretion of organic cations (OCs). Tributylmethylammonium (TBuMA; mol wt 200) and triethylmethylammonium (TEMA; mol wt 116) were selected as model OCs that exhibit significant and negligible biliary excretion, respectively, in rats. The apparent lipophilicity of TBuMA, but not that of TEMA, was increased by the presence of either rat bile or specific bile salts, suggesting the formation of lipophilic ion pair complexes for TBuMA with bile salts in the liver. The uptake of TBuMA into canalicular liver plasma membrane (cLPM) vesicles, but not that of TEMA, was increased in the presence of bile salts, with a significant increase for both ATP-dependent transport and passive diffusion. The uptake of TBuMA in the presence of the bile salts was inhibited by representative P-glycoprotein (P-gp) substrates and vice versa, suggesting the involvement of P-gp in the canalicular excretion of TBuMA-bile salt complexes in vivo. Increased affinity toward P-gp is suggested as the mechanism responsible for the increased ATP-dependent transport for the ion pair complexes. We propose that ion pair formation with bile slats in hepatocytes may be responsible for the preferential biliary excretion of high-molecular-weight OCs including TBuMA.