Folate conjugase activities and folate concentrations in blood obtained from patients with chronic renal failure and healthy controls were measured by radioassay using pteroyldiglutamyl-[14C]-glutamic acid as substrate and microbiologic assay, respectively. A total of 32 patients receiving hemodialysis for an average of 5.4 years participated in the study. Folate conjugase activities in posthemodialysis plasma were significantly higher than those in prehemodialysis samples and were similar to those of controls. Plasma folate conjugase was inhibited by the in vitro addition of the heated extract of prehemodialysis plasma. The heated extract of urine from a healthy control also inhibited the enzyme. Furthermore, plasma folate conjugase activities were reduced by the in vitro addition of sulfate, one of the major constituents in urine, although the addition of urea or uric acid had no such effect. Data indicate the presence of an unidentified heat-stable inhibitor(s) that is excreted by the normal kidney, and sulfate may be one of them. During hemodialysis, plasma folate concentrations decreased, while erythrocyte folate concentrations remained unchanged. Five of 32 patients demonstrated plasma folate concentrations lower than normal. These findings suggest that a careful evaluation of folate requirements is necessary in patients maintained with hemodialysis. © 1994.