Milk fat was investigated in lactating dairy cows fed diets supplemented with Ca salts of trans fatty acids (Ca-tFA) or Ca salts of conjugated linoleic acids (Ca-CLA). Forty-five Holstein cows ( 115 days in milk) were fed a control diet (51% forage; dry matter basis) supplemented with 400 g of EnerG II ( Ca salts of palm oil fatty acids) for 2 wk; subsequently, 5 groups of 9 cows each were assigned for 4 wk to the control diet or diets containing 100 g of Ca-CLA or 100, 200, or 400 g of Ca-tFA in a randomized block design. Treatments had no effect on dry matter intake, milk production, protein, lactose, or somatic cell count. Milk fat percentage was reduced from 3.39% in controls to 3.30, 3.04, and 2.98%, respectively, by the Ca-tFA diets and to 2.54% by the Ca-CLA diet. Milk fat yield (1.24 kg/d in controls) was decreased by 60, 130, and 190 g/d with increasing dose of Ca-tFA and by 290 g/d with the Ca-CLA supplement. Consistent with increased endogenous synthesis of cis-9-containing CLA from precursors provided by the Ca-tFA diets, total CLA were similar in milk of cows fed Ca-CLA or Ca-tFA. Compared with controls, the Ca-CLA diet increased trans-10, cis-12-18: 2 yield in milk, without altering levels of trans-18: 1 isomers. In contrast, yields of most trans-18: 1 isomers were elevated in milk of cows fed Ca-tFA diets, whereas yields of trans-10, cis-12-18: 2 remained similar to control values. We conclude that milk fat depression can occur without an increase in trans-10, cis-12-18: 2 in milk and that other components, perhaps the trans-10-18:1 isomer, may be involved.