The extrarenal synthesis of 1,25-dihydroxyvitamin D [1,25-(OH)2D] is a characteristic of activated macrophages and has been demonstrated to occur in vitro in synovial fluid macrophages from patients with inflammatory arthritis. To examine whether such synthesis occurs in vivo, 19 patients with rheumatoid arthritis, 5 patient controls, and 5 healthy controls received a challenge oral dose of 250 micrograms 25-hydroxyvitamin D3 (25-OHD3) and the serum 1,25-(OH)2D3 response was measured. The median rise in serum 1,25-(OH)2D3 was significantly greater (22 pg/ml) in the rheumatoid patients compared to either of the control groups (8 pg/ml), although the increase in precursor 25-OHD3 was similar in all groups. The serum 1,25-(OH)2D concentration did not rise above the normal upper limit in any of the control subjects but exceeded the normal range in 8 of the rheumatoid patients. Extrarenal 1,25-(OH)2D synthesis is substrate dependent, unlike renal 1 alpha-hydroxylation, which is homeostatically controlled. Excessive 1,25-(OH)2D3 synthesis in the rheumatoid group on raising the 25-OHD3 concentration is indicative of nonrenal production of the hormonal metabolite. Further evidence for substrate-dependent extrarenal synthesis came from measurements of 25-OHD and 1,25-(OH)2D in paired serum and synovial fluid samples from 19 patients with inflammatory arthritis, including 15 with rheumatoid arthritis. Synovial fluid 1,25-(OH)2D was usually present at a lower concentration than serum 1,25(OH)2D, with which it was strongly correlated (Kendall's R = 0.46, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)