As previously reported, the association of bone loss with an increase in bone marrow adipose volume may be related to the inhibition of human osteoblastic cell proliferation in the presence of human adipocytes. In the osteoblastic supernatant, fatty acid composition varied after coculture with mature adipocytes, with a marked increase in the proportion of docosahexaenoic acid (22:6 n-3; DHA) (+90 +/- 8%). This suggests that polyunsaturated fatty acids (PUFA) may contribute to the inhibitory effect of adipocytes on osteoblastic cell proliferation. The purpose of the present study was to evaluate the effects of two PUFA, DHA and arachidonic acid (20:4 n-6; AA), on the proliferation of primary human osteoblastic (hOB) cells and human osteosarcoma cell line, MG-63. The effects of cholesterol and oleic acid, a monounsaturated FA (18:1 n-9; OA), both being present in adipocyte lipidic vacuoles, were also investigated. At between 10 and 50 micromol/L, DHA and AA induced a significant dose-dependent decrease in hOB cell proliferation (p < 0.0001 and p < 0.006 for DHA and AA, respectively) when compared with control hOB cells exposed to the vehicle (bovine serum albumin). This inhibition reached -50% with 50 micromol/L of DHA or 20 micromol/L of AA. This effect was not related to cell apoptosis, as shown by terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end labeling (TUNEL) and Hoechst dye staining. In contrast, OA and cholesterol had no effect on hOB cell proliferation, even at a high concentration (200 micromol/L). Similar results were observed with regard to MG-63 cell proliferation. In addition, flow cytometric analysis showed that the number of hOB cells in the S phase of the cycle was twofold lower when treated with 50 micromol/L of DHA or AA. In vitro results indicate that mature adipocytes may contribute to age-related bone loss through the release of polyunsaturated fatty acids, which impair osteoblastic proliferation.