The objective of this study was to examine the effect of macrolide antibiotics, clarithromycin, erythromycin, roxithromycin, josamycin and azithromycin, on the hepatic uptake of digoxin. The uptake of [3H]digoxin was studied in rats in vivo, using the tissue-sampling single-injection technique, and in isolated rat hepatocytes in vitro. The uptake of [3H]digoxin into rat hepatocytes was concentration-dependent with a Michaelis constant (Km) of 445 nM. All the macrolide antibiotics inhibited the uptake of [3H] digoxin into rat hepatocytes in a concentration-dependent manner. However, clarithromycin did not affect the in vivo hepatic uptake of digoxin in rats. The in vivo permeability-surface area product of digoxin for hepatic uptake (PSinf) was estimated to be 12.5ml/min/g liver from the present in vitro data, which is far larger than the hepatic blood flow rate (1.4ml/min/g liver). Macrolide antibiotics at clinically relevant concentrations inhibit digoxin uptake by rat hepatocytes in vitro, but not in vivo, probably because hepatic uptake of digoxin in rats is blood flow-limited. Clinically observed digoxin-macrolide interaction in humans could be due to macrolide inhibition of hepatic digoxin uptake, if the uptake is permeation-limited.