Cyclooxygenase (COX) catalyses the rate-limiting step of prostanoid biosynthesis. Two COX isoforms have been identified, COX-1, the constitutive form, and COX-2, the inducible form. While COX-2 has been implicated in body fat regulation, the underlying cellular mechanism remains to be elucidated. The present study was undertaken to examine the potential role of COX in modulating adipogenesis and to dissect the relative contribution of the two isoenzymes in this process. COX-2 was found to be expressed in undifferentiated 3T3-L1 cells and down-regulated during differentiation, whereas the cellular level of COX-1 remained relatively constant. Abrogating the activity of either of these two isoenzymes by selective COX inhibitors accelerated cellular differentiation, suggesting that both COX isoenzymes negatively influenced differentiation. Tumor necrosis factor-alpha (TNFalpha) significantly up-regulated COX-2 expression ( approximately 2-fold) in differentiating 3T3-L1 cells, whereas similar effect was not observed with COX-1 expression. Abrogating the induced COX-2 activity reversed the TNFalpha-induced inhibition of differentiation by approximately 70%, implying a role for COX-2 in mediating TNFalpha signaling. Hence, both COX isoforms were involved in the negative modulation of adipocyte differentiation. COX-2 appeared to be the main isoform mediating at least part of the negative effects of TNFalpha.