Hemodialysis patients have a capacity for extrarenal production of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]; however, the source of the hormone is unknown in these patients. Since 1,25(OH)2D3 synthesis by cultured hematopoietic cells has been demonstrated previously, we assessed hormone production by mononuclear cells from peripheral blood obtained from normal subjects (n = 6), uremic patients not yet requiring dialysis (n=4) and hemodialysis patients (n = 14). 1,25(OH)2D3 production was analyzed by sequential straight phase and reverse phase HPLC. In the hemodialysis group, the mean specific production of a metabolite co-eluting with 1,25(OH)2D3 (in fmol/100,000 cells/h) both by monocyte-enriched adherent cells (Mo) and lymphocyte-enriched non-adherent cells (Ly) was increased as compared to non-dialyzed subjects (119 vs. 22 for Mo, not significant, 65 vs. 14 for Ly, p < 0.05). Taken together, Mo and Ly from hemodialysis patients synthesized significantly more 1,25(OH)2D3 (p < 0.02) than non-dialyzed subjects (184 vs. 36, means). No differences were found between cells from normal subjects and patients with preterminal renal failure. Exposure of cultured normal Mo (n = 6) to cuprophane (CU), polyacrylonitrile (AN69) or polycarbonate-polyether (PC) membrane devices resulted in increased 1,25(OH)2D3 CU, whereby only PC (p < 0.05) was significantly different from control. Our results suggest that blood mononuclear cells contribute to extrarenal 1,25(OH)2D3 synthesis in hemodialysis patients, and that this synthetic activity may be related to the hemodialysis procedure