Proinflammatory cytokines such as tumor necrosis factor (TNF) alpha are well known to inhibit adipocyte differentiation. TNF-alpha triggers ceramide synthesis through binding of TNF-alpha to its p55 receptor. Therefore, ceramide is implicated in many of the multiple signaling pathways initiated by TNF-alpha. In breast tissue engineering, it is important to know how to modulate adipocyte differentiation of the stem cells with exogenous additives like ceramide in vitro. We hypothesized that stem cell adipogenesis could be retained in TNF-alpha-treated preadipocytes in which ceramide synthesis was blocked and that exogenous ceramide could inhibit adipocyte differentiation. We first studied the effect of ceramide synthase inhibitor, Fumonisin B2, on the adipogenesis of murine mesenchymal stem cells (D1 cells), treated with TNF-alpha. We then studied the effect of specific exogenous C6-ceramide on D1 cell viability and differentiation. It was found that 1 ng/ml of TNF-alpha significantly inhibited D1 cell adipogenesis. Cells treated with 5 mu M of Fumonisin B2 were able to undergo adipogenesis, even when treated with TNF-alpha. High concentrations of exogenous C6-ceramide (> 50 mu M) had an inhibitory effect, not only on the pre-confluent proliferation of the D1 cells but also on the post-confluent cell viability. High concentrations of C6-ceramide (> 50 mu M) also inhibited mitotic clonal expansion when D1 cell differentiation was induced by the addition of an adipogenic hormonal cocktail. C6-ceramide at low concentrations (10-25 mu M) inhibited lipid production in D1 cells, demonstrated by decreased levels of both total triglyceride content and specific fatty acid composition percentages. Genetic expression of peroxisome proliferator-activated receptor (PPAR)gamma and aP2 in D1 cells was reduced by C6-ceramide treatment. CCAAT/enhancer-binding protein (C/EBP) beta levels in D1 cells were reduced by C6-ceramide treatment during early differentiation; PPAR. and aP2 protein levels were reduced at terminal differentiation. C6-ceramide at lower concentrations also decreased lipid accumulation of differentiating D1 cells. Our results suggest that ceramide synthase inhibitor retains the adipogenic potential of TNF-alpha-treated mesenchymal stem cells, while exogenous ceramide at lower concentrations inhibit the adipogenesis of mesenchymal stem cells. Ceramide, therefore, could be a modulator candidate in breast tissue engineering strategies.