This study was designed to investigate the effects of three diets with different levels of trans fatty acids and the physiologic status on the physicochemical properties and enzymatic activities of liver microsomes and mitochondria. Three groups of 10 female weaning rats each were fed for 10 wk one of three diets differing in their trans fatty acid contents (Control, 0 mol/100 mol total fatty acids; high, 14.5 mol/100 mol; very high, 30 mol/100 mol). At the onset of adult life (10 wk of age), they were mated. Six rats in each group were killed at the end of gestation (Pregnant rat groups). The four remaining pregnant rats continued to receive their experimental diets until weaning of their litters. Six pups from the litters for each group (3 males and 3 females) were selected and fed the same experimental diet as the dams from wk 3 to 10 of age (2nd generation virgin groups) and then killed. Trans fatty acid levels in liver microsomes and mitochondria rose in parallel with the dietary trans fatty acid content, whereas saturated fatty acids dropped in both organelles with increasing trans fatty acids. Pregnant and 2nd generation adult rats fed trans isomers also had lower levels of cholesterol and a lower cholesterol/phosphorus ratio in their liver microsomes compared with controls. A significant interaction between diet and pregnancy was detected in the activities of delta6-desaturase and glucose-6-phosphatase in liver microsomes. Dietary trans fatty acids decreased the activities of both enzymes but only in pregnant rats. No differences in the fluorescence anisotropy of membranes or the enzymatic activities in liver mitochondria were observed. In conclusion, dietary trans fatty acids significantly lowered cholesterol and the cholesterol/phosphorus ratio in liver microsomes. This effect might contribute to low delta6-desaturase and glucose-6-phosphatase activities in liver microsomes of pregnant rats.