Osteoblasts in culture can differentiate into mature mineralizing osteoblasts when stimulated with osteogenic agents. Clinical trials and in vivo animal studies suggest that specific polyunsaturated fatty acids (PUFAs) may benefit bone health. The aim of this study was to investigate whether arachidonic acid (AA) and docosahexaenoic acid (DHA) affect osteogenesis in osteoblasts and the transdifferentiation into adipocytes. Results from this study show that long-term exposure to AA inhibited alkaline phosphatase (ALP) activity in these cells, which might be prostaglandin E2 (PGE2)-mediated. DHA exposure also inhibited ALP activity which was evident after both short- and long-term exposures. The mechanism whereby DHA inhibits ALP activity is not clear and needs to be investigated. Although long-term exposure to PUFAs inhibited ALP activity, the mineralizing properties of these cells were not compromised. Furthermore, PUFA exposure did not induce adipocyte-like features in these cells as evidenced by the lack of cytoplasmic triacylglycerol accummulation. More research is required to elucidate the cellular mechanisms of action of PUFAs on bone. Copyright © 2008 John Wiley & Sons, Ltd.