Trans-10, cis-12 conjugated linoleic acid (CLA) is a potent inhibitor of milk fat synthesis. We examined the effect of low doses of trans-10, cis-12 CLA using Holstein cows in a 4 x 4 Latin square design. Milk yield and milk protein were unaffected, but abomasal infusion of 1.25, 2.5, and 5.0 g/d of trans-10, cis-12 CLA reduced milk fat yield by 7, 16, and 29%, respectively. When combined with previous data, the reduction in milk fat yield was curvilinear, relating to both quantity infused and milk fat content of trans-10, cis-12 CLA (R2 = 0.99 and 0.96, respectively). Further, changes in milk fatty acid composition indicated the mechanism involved inhibition of de novo fatty acid synthesis and the utilization of circulating fatty acids.