Under basal conditions, bile salt secretion by the liver is not affected by microtubule disruption. However, when a bile salt load is imposed on the liver, a microtubule-dependent secretion mechanism is recruited (J. Lipid Res. 1988. 29: 144-156). We tested the hypothesis that recruitment of this microtubule-dependent mechanism is influenced by the relative hydrophobicity of the bile salts being secreted. Intact male rats were depleted of bile salts by overnight biliary diversion, pretreated with colchicine (a microtubule inhibitor) or its inactive isomer, lumicolchicine (control), and reinfused intravenously with bile salts of increasing hydrophobicity (taurodehydrocholate < tauroursodeoxycholate < taurocholate) at 200 nmol/min.100 g. After 45 min, when steady-state bile salt secretion was achieved, tracer [3H]taurocholate was administered intravenously. The colchicine-insensitive component of bulk bile salt secretion was constant at approximately 130 nmol/min.100 g, and the colchicine-sensitive component increased from approximately 0 to 35 and 60 nmol/min.100 g, respectively, with reinfusion of the more hydrophobic bile salts. Retained bile salts accumulated in the liver and serum and were detectable in urine. Peak biliary secretion of [3H]taurocholate in control animals increased linearly from 15.3 to 18.0% administered dose/min with increasing hydrophobicity of the secreted bile salts (P < 0.002). In colchicine-pretreated animals, peak secretion rates decreased linearly from 13.8 to 9.2%/min (P < 0.001), with maximal inhibition in taurocholate-reinfused animals (P < 0.01). Utilization of a microtubule-dependent secretion mechanism increases with increasing bile salt hydrophobicity. This mechanism permits more efficient hepatic secretion of bile salts, but increases the susceptibility of bile salt secretion to microtubule disruption. We postulate that microtubule-dependent insertion of bile salt transporters into the canalicular membrane underlies the enhanced bile salt secretion observed when a bile salt load is imposed upon the liver.