Both vitamin D and inflammatory cytokines can stimulate osteoclast formation and activity. We studied the effect of 1,25-dihydroxycholecalciferol (1,25(OH)2D), and interleukin-6 (IL-6), on the formation and activity of feline osteoclasts, using peripheral blood mononuclear cells (PBMCs) from cats with and without tooth resorption (TR+ and TR−) as a source of osteoclast precursors. The formation of osteoclast-like cells (defined as multinucleated, tartrate-resistant acid phosphatase-positive cells) was assessed at 7 and 14days. In the presence of M-CSF and RANKL, with and without IL-6, more osteoclasts were formed from TR− PBMCs than from TR+ PBMCs on plastic. More osteoclasts were formed from TR+ PBMCs on bone slices in the presence of M-CSF/RANKL with 1,25(OH)2D. This opposite effect may be due to a higher expression of the vitamin D receptor (VDR) in TR+ osteoclasts and precursors on bone. Formation of resorption pits was analyzed and confirmed with scanning electron microscopy. In conclusion, we propose that TR+ PBMCs when cultured on bone are sensitive to 1,25(OH)2D, whereas the differentiation of TR− PMBCs on bone seem more sensitive to IL-6, suggesting that osteoclast precursors from cats with and without tooth resorption respond differently to osteoclast stimulating factors.