Cobalamin (Cbl; vitamin B12) malabsorption in pancreatic insufficiency can be partially corrected by bicarbonate and completely corrected by pancreatic proteases but the mechanisms involved are unknown. Because saliva contains enough R type Cbl binding protein (R protein) to bind all of the dietary and biliary Cbl, it is possible that R protein acts as an inhibitor of Cbl absorption and that pancreatic proteases are required to alter R protein and prevent such inhibition. To test this hypothesis we studied the ability of R protein and intrinsic factor (IF) to compete for Cbl binding and ability of pancreatic proteases to alter this competition. Human salivary R protein bound Cbl with affinities that were 50 and 3 fold higher than those of human IF at pH 2 and 8, respectively. Cbl bound to IF was transferred to an equal amount of R protein with t(1/2)'s of 2 and 90 min at pH 2 and 8, respectively, and within several hours respective ratios of R protein Cbl/IF Cbl of 50 and 2 were observed. Cbl bound to R protein was not transferred to IF at either pH 2 or 8. Incubation of R protein with pancreatic proteases at pH 8 led to a 150 fold decrease in its affinity for Cbl. Incubation of R protein Cbl with pancreatic proteases led to complete transfer of Cbl to IF within 10 min. Gel filtration studies with R protein [57Co]Cbl and 125I R protein showed that pancreatic proteases partially degraded R protein. Pancreatic proteases differed in their ability to effect these changes with trypsin > chymotrypsin > elastase. Pancreatic proteases did not alter IF in any of the parameters mentioned above. Pepsin failed to alter either R protein or IF. These studies suggest the following: (a) that Cbl is bound almost exclusively to R protein in the acid milieu of the stomach, rather than to IF as has been assumed previously; (b) that Cbl remains bound to R protein in the slightly alkaline environment of the intestine until pancreatic proteases partially degrade R protein and enable Cbl to become bound exclusively to IF; and (c) that the primary defect in Cbl absorption in pancreatic insufficiency is a lack of pancreatic proteases and a failure to alter R protein and effect the transfer of Cbl to IF. These studies also suggest that the partial correction of Cbl malabsorption observed with bicarbonate is due to neutralization of gastric HCl, since at slightly alkaline, pH IF can partially compete with R protein for the initial binding and retention of Cbl.