It has been suggested that bacteria in the intestine gain access into the biliary tract by entering the portal‐venous blood. We have tested the hypothesis of hematogenous infection of the biliary system in cats. The animals were treated in three different groups: group A (no biliary obstruction), group B (acute biliary obstruction) and group C (chronic biliary obstruction). A mutant strain of Escherichia coli was infused into the splenic vein of cats at three different dosages (10⁷, 10⁵ and 10³) with sham controls. In the unobstructed biliary system, the mutant E. coli was isolated from the bile 30 min and 90 min after the infusion of 10⁷ and 10⁵ E. coli, respectively. No bacteria were found in the bile with the infusion of 10³ E. coli and in the control animals. Bile flow was significantly reduced with the infusion of bacteria. The biliary excretion of E. coli in group B was similar to that in group A. In group C, the bile output in the first hour was very high but declined rapidly. E. coli was excreted into the bile at all three dosages of infusion after 30 min. Histological sections of the liver showed that the infused bacteria entered the sinusoidal blood and that some were phagocytosed by Kupffer cells. The portal‐venous blood was considered an important route of bacterial invasion into the biliary system, and the penetration of bacteria was facilitated in biliary obstruction. (HEPATOLOGY 1991;14:313–317.)