The distribution and metabolism of secretin and aprotinin were studied in isolated perfused rat pancreas, which were kept under physiological conditions. At 125I-[Tyr1]secretin perfusion study the radioactivity appeared in the effluent, rapidly attaining an equilibrium with the perfusate. While the intact secretin concentration decreased to 55% of the perfusate; the distribution space of secretin was estimated to be about 0.11 ml/g pancreas. The ratio of intact secretin to degradated products in the pancreas increased concomitantly with the perfusion of 82 nM secretin. When unlabeled secretin was perfused, immunoreactive secretin concentration in the portal effluent reached also a rapid equilibrium to about 60% of the perfusate. These results suggested that pancreas might clear about 40% of the entering secretin. In the case of 125I-aprotinin perfusion, intact aprotinin fraction was predominant both in the effluent and in the pancreas. The apparent distribution space was about 40% of the volume of pancrease. Meanwhile, when 125I-reduced aprotinin or 125I-[S-carboxamidomethyl]aprotinin was perfused, the decomposed products were significantly increased. These findings indicate that the disulfide bonds in aprotinin molecule play an important role for its stability as well as its antiproteolytic activity.