Previous work had shown that dietarytrans fatty acids (tFA) resulted in decreased fat deposition in adipose tissue. This study was conducted to see iftFA influence lipid accumulation in Swiss mouse fibroblast 3T3-L1 cells, which are widely used as an adipocyte model. Cells were cultured in the presence of experimental or control growth media supplemented with fatty acids complexed to bovine serum albumin. Fatty acid compositions of experimental and control growth media were similar except that the octadecenoates in the control growth media werecis fatty acids, whereas those in the experimental media contained bothcis andtrans fatty acids. Cell-conditioned media and cellular lipids at the preadipocyte and differentiating adipocyte stages were analyzed. At both stages of development, less fat accumulated, in cells cultured in the presence oftFA, due primarily to a decrease in the nonpolar lipid content of cells exposed totFA, and linoleate to arachidonate ratios were higher in cells supplemented withtFA. Calculations comparing sums of saturated and monounsaturated fatty acids in cells at the differentiating adipocyte stage suggested thattFA may have replaced monoun-saturated fatty acids in the nonpolar lipid fraction and saturated fatty acids in the polar lipid fraction. The results of these studies are in good agreement with thein vivo effects oftFA seen in previous work with mouse adipose tissue. It was concluded that the 3T3-L1in vitro model is an appropriate system for further studies oftFA and lipid metabolism in adipose tissue.