Cis 9, trans 11 (c 9, t11)-18: 2 and trans 10, cis 12 (t10, c12)-18: 2 are the major conjugated linoleic acid (CLA) isomers in dietary supplements which reduce milk fat content in nursing women. The present study evaluated the effects of each CLA isomer or vaccenic acid on body composition and tissue fatty acids during lactation in mice. Dams were fed 30 g rapeseed oil (control)/kg diet or 20 g control plus 10 g 18: 0, trans 11–18: 1 (t11–18: 1), c 9, t11–18: 2, or t10, c12–18: 2. Dietary t10, c12–18: 2 reduced food intake by 18 % and carcass fat weight of the dams by 49 % compared with the other treatments. Milk fat percentage ranked by treatment was 18: 0>t11–18: 1=c 9, t11–18: 2>t10, c12–18: 2. The sum of saturated 12: 0 to 16: 0 in milk fat was lower when c 9, t11–18: 2 was fed compared with the control, 18: 0, or t11–18: 1 treatments. Dietary t10, c12–18: 2 caused further reductions in milk fat 12: 0 to 16: 0. The proportion of CLA isomers was 3-fold greater in milk fat than in the carcasses of the dams. The pups nursing from the dams fed t10, c12–18: 2 had the lowest body weights and carcass fat, protein, and ash contents. Nursing from the dams fed c 9, t11–18: 2 also resulted in lower carcass fat compared with the 18: 0 or t11–18: 1 treatments. The ratios of cis 9–16: 1:16: 0 or cis 9–18: 1:18: 0, proxies for Δ9-desaturase activity, were markedly lower in the carcasses of the dams and pups fed t10, c12–18: 2. The ratio of 20: 4n-6:18: 2n-6, a proxy for Δ6- and Δ5-desaturase and elongase activity, in the liver of the dams and pups fed t10, c12–18: 2 also was lower. Dietary t11–18: 1 enhanced the content of c 9, t11–18: 2 in milk fat and carcasses. As in previous studies, the reduction in food intake by t10, c12–18: 2 could not entirely account for the marked decrease in carcass fat content and milk fat concentration. T10, c12–18: 2 probably had a negative effect on Δ9-desaturase and mammary de novo fatty acid synthesis. Although these effects need to be confirmed in lactating women, the results suggest that the consumption of supplements containing t10, c12–18: 2 should be avoided during the nursing period.