The binding of pirenzepine, a selective muscarinic receptor antagonist to plasma and bile, was studied in vitro and in vivo. Plasma and hepatic bile were incubated with 14C-pirenzepine and the bound fraction of 14C-pirenzepine determined by equilibrium dialysis. The bound fractions were 12.6% (s.e.m. = 1.5) and 12.1% (s.e.m. = 1.6) in plasma and bile samples, respectively. After in vitro incubation, the radioactivity in both plasma and bile was removed by exhaustive dialysis against water for up to 94 h, suggesting that the binding was a noncovalent association. 14C-Pirenzepine was also given intravenously to five postoperative patients with T-tube drainage of hepatic bile. In plasma, 12.6% (s.e.m. = 1.8) of 14C-pirenzepine was reversibly bound to albumin. By contrast, in bile 13.4% (s.e.m. = 3.2) was irreversibly bound, mainly to bilirubin glucuronides (greater than 90%). After an intravenous injection of 14C-pirenzepine, the radioactivity in plasma decreased bi-exponentially with an initial distribution half-life of 0.24 h and an elimination half-life of 10.2 h. The radioactivity reappeared cyclically in bile, suggesting enterohepatic recirculation of 14C-pirenzepine.