Milk fat from Jersey cows contains less oleic acid (cis-C-18:1) and more short- and medium-chain fatty acids than does milk fat from Holstein cows. The objective of this experiment was to determine responses in milk fat composition when Jersey and Holstein cows were fed diets either high (37% of dry matter) or low (27% of dry matter) in content of nonstructural carbohydrates (NSC) and supplemented with either 0 or 2.5% (of dry matter) of a mostly saturated fat source. Four Holstein cows and four Jersey cows were used in a Latin square design with 28-d periods; diets were in a 2 x 2 factorial arrangement. Fat supplementation decreased contents of fatty acids synthesized de novo within the mammary gland and increased contents of C-18:0 and cis-C-18:1. Low-NSC diets tended to increase C-16:0 and to decrease C-18:0, cis-C-18:1, and C-18:3 Despite the differences in fatty acid composition between breeds, both breeds generally responded similarly to dietary treatments. An interaction of breed and fat indicated that the content of CiS-C-18:1 in milk fat was increased more by supplemental fat in Holsteins than in Jerseys. Interactions of breed x fat and breed x carbohydrate type showed that the ratio of cis-C-18:1 to C-18:0 decreased when Jerseys were supplemented with fat but increased for Holsteins, and decreased when Jerseys were fed the low-NSC diet but increased when Holsteins were fed low NSC. The data are consistent with the hypothesis (Beaulieu and Palmquist, 1995, J. Dairy Sci. 78:1336-1344) that mammary activity of stearoyl-coenzyme A desaturase is lower in Jerseys than in Holsteins.