The purpose of these studies was to define the pathways by which bacteria pass from bile duct to bloodstream during acute bacterial cholangitis in the rat. The respective roles of biliary obstruction and intrabiliary pressure during the reflux of biliary bacteria were defined by the infusion of bacteria via the bile duct into rats with or without prior bile duct obstruction. As determined by quantitative blood culture analysis, bacterial reflux from bile to blood was enhanced by increased intrabiliary pressure regardless of presence or absence of biliary obstruction. Light microscopic examination of rat liver 48 hours after bile duct obstruction revealed bile ductular proliferation and bile canalicular dilatation. Light microscopic autoradiographs showed aggregates of tritiated thymidine-labeled Escherichia coli outside of interlobular bile ducts in the portal tracts. Transmission electron microscopic examination of rat liver perfused with a bacterial suspension via the common bile duct showed disruption of liver cells and formation of intracellular vacuoles. Bacteria appeared to enter the sinusoidal spaces via these intracellular vacuoles. We conclude that during retrograde biliary infusion (1) increased intrabiliary pressure is the main determinant of increased bacterial reflux into blood; (2) bacteria enter the bloodstream by predominantly intracellular pathways; and (3) prior biliary obstruction is not a significant factor in bacterial reflux from bile to bloodstream.