Vitamin D is an important regulator of mineral homeostasis and bone metabolism. 1-Hydroxylation of 25-(OH)D₃ to form the bioactive vitamin D hormone, 1,25-(OH)₂D_3, is classically considered to take place in the kidney. However, 1-hydroxylase has been reported at extrarenal sites. Whether bone is a 1,25-(OH)₂D₃ synthesizing tissue is not univocal. The aim of this study was to investigate an autocrine/paracrine function for 1,25-(OH)₂D₃ in bone. We show that 1-hydroxlase is expressed in human osteoblasts, as well as the vitamin D binding protein receptors megalin and cubilin. Functional analyses demonstrate that after incubation with the 1-hydoxylase substrate 25-(OH)D_3, the osteoblasts can produce sufficient 1,25-(OH)₂D₃ to modulate osteoblast activity, resulting in induced alkaline phosphatase (ALP) activity, osteocalcin (OC) and CYP24 mRNA expression, and mineralization. The classical renal regulators of 1-hydroxylase, parathyroid hormone, and ambient calcium do not regulate 1-hydroxylase in osteoblasts. In contrast, interleukin (IL)-1 strongly induces 1-hydroxylase. Besides the bone-forming cells, we demonstrate 1-hydroxylase activity in the bone resorbing cells, the osteoclasts. This is strongly dependent on osteoclast inducer RANKL. This study showing expression, activity, and functionality of 1-hydoxylase unequivocally demonstrates that vitamin D can act in an auto/paracrine manner in bone.--van Driel, M., Koedam, M., Buurman, C. J., Hewison, M., Chiba, H., Uitterlinden, A. G., Pols, H. A. P., van Leeuwen, J. P. T. M. Evidence for auto/paracrine actions of vitamin D in bone: 1-hydroxylase expression and activity in human bone cells.