The osteopenic bone disease associated with primary biliary cirrhosis is thought to be de to a deficiency in vitamin D or its metabolites. However, this has never been proven. Therefore, we measured serum levels of 25-hydroxyvitamin D3 (25-OHD3), 1,25-dihydroxyvitamin D (1,25(OD)2D), and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), before and after a 1 yr treatment with oral 25,OHD3, in 10 patients with primary biliary cirrhosis selected because of documented osteopenic bone disease. Only in 1 patient was the pretreatment serum 25-OHD3 level below normal, less than 4.4 ng/ml. In 8 patients the serum 25,OHD3 level was in the low normal range and in one, was above normal. Serum levels of 1,25(OH)2D, the vitamin D metabolite with the greatest stimulatory effect on intestinal calcium absorption, were normal in 9 patients and elevated in 1. In contrast, serum levels of 24,25-(OH)2D3, a metabolite whose function is not known with certainty, were undetectable in 8 patients, low normal in a ninth, and normal in 1 patient who had been on large amounts of vitamin D2 (50,000 U b.i.w.,) before the start of the study. After 1 yr of treatment with oral 25-OHD3, serum 25-OHD3 rose to above normal in 9 patients. Serum 1,25-(OH)2D levels did not change significantly, while 24,25-(OH)2D3 rose to normal levels or higher in 9 of 10 patients. The bone disease of primary biliary cirrhosis is not due to 25-hydroxyvitamin D deficiency alone and is certainly not due to a deficiency of 1,25-(OH)2D as has been postulated. It may be related to low blood levels of 24,25-(OH)2D3 or to other as yet undefined factors.