Substrate films of starch, RNA, and DNA were used to identify the isoenzymes of amylase, RNAase, and DNAase found in human ductal pancreatic juice subjected to isoelectric focusing. The pancreatic secretions from 15 patients were shown to contain as many as four isoenzymes of RNAase; the two major forms had isoelectric points of 7.87 and 7.52, and the two minor forms, of 7.25 and 6.90. Six DNAase bands were detected; the major bands had pI values of 4.86 and 4.79, and sometimes appeared as one band. The minor bands had pI values of 5.08, 5.00, 4.68, and 4.58. Purified bovine DNAase I, analyzed similarly, showed four bands (5.29, 5.19, 5.04 and 4.96). Nine isoenzymes of alpha-amylase were observed in the secretions from 15 patients. The major alpha-amylase isoenzyme had a pI value of 6.84 in 14 patients and of 7.04 in 1 patient. Secondary bands were seen with pI values of 6.23, 6.53 and 6.69. Additional isoenzymes were found with pI values of 7.16, 6.39, 6.00 and 5.78. The amylase isoenzyme with a pI value of 6.39 was found in 7 of the 8 patients with a normal pancreas or carcinoma of the pancreas, and in only 1 of 7 patients with acute or chronic pancreatitis.