Because of the suggestion that intestinal alkaline phosphatase was elevated in the serum of patients with chronic renal failure, we studied the serum of 42 patients undergoing hemodialysis with elevated enzyme activity. Using a sensitive and specific electroimmunoassay for the intestinal isoenzyme, 26 of 42 serum samples were positive, compared with 3 of 25 samples obtained from hospitalized patients with elevated phosphatase activity. The fractional amount of this isoenzyme was also higher, ranging from 1.5% to 41% of the total serum phosphatase, compared with 0.1%-1.2% in control sera. Kidneys removed during transplantation or postmortem contained a membranous phosphatase with immunologic activity identical to the intestinal isoenzyme in 5 of 6 patients. This enzyme accounted for 8%-21% of the total kidney phosphatase activity. By morphology the immunoreaction was localized to the apical membranes of the collecting tubules. Thus, the kidney is the likely source of the observed increase in serum intestinal-type phosphatase activity noted in patients with chronic renal failure. An elevation in the intestinal isoenzyme rather than the presence of early metabolic bone disease or hepatic disease should be considered in renal failure patients with mildly elevated (up to 50% over normal) total serum alkaline phosphatase.