Fibrinolytic properties have been detected in animal and human gallbladder (GB) bile. Plasminogen activator inhibitor-1 (PAI-1) has been reported in greater concentration in GB stone bile and may be a nucleating factor in the pathogenesis of GB stone formation. It is unknown whether or not human choledochal bile has similar properties, which could have a role in choledocholithiasis. The aims of this study were to determine the presence of fibrinolytic properties of human choledochal bile and to compare those properties among normal, acalculous, and calculous-infected choledochal bile. Tissue plasminogen activator (t-PA) and PAI-1 of choledochal bile were measured by enzyme linked immunosorbent assay in patients with cholangitis due to acalculous bile duct obstructions (n = 9), choledocholithiasis with cholangitis (n = 20), and normal bile (n = 7). The t-PA concentration of choledochal bile was no different among the three groups (acalculous-infected bile, median 4.61 ng/ml, and calculous-infected bile, 4.61 ng/ml, versus normal bile, 7.33 ng/ml). PAI-1 was detected in choledochal bile in significantly greater concentrations in patients with acalculous cholangitis due to bile duct obstructions and choledocholithiasis with cholangitis (acalculous-infected bile, median 0.36 ng/ml, and calculous-infected bile, 0.1 ng/ml, versus normal bile, 0.02 ng/ml, p < 0.05), but the bile concentration of PAI-1 was no different between the acalculous and calculous-infected choledochal bile. Human choledochal bile possesses t-PA and PAI-1. PAI-1 was present in greater concentrations in both acalculous and calculous-infected choledochal bile. Increased levels of PAI-1 may be an epiphenomenon of cholangitis rather than a factor in the pathogenesis of choledocholithiasis.