Changes in mammary AA utilization associated with dietary fat-induced milk protein depression were assessed in cows with disturbed AA status. Four first lactation cows fitted with rumen cannulas were assigned to a factorial arrangement of treatments: two diets and two casein infusion sites within a 4 x 4 Latin square design. Diets were formulated with yellow grease at 0 or 4% of DM. An 8% sodium caseinate solution was infused continuously at 5.04 kg/d into the rumen or abomasum during the last 5 d of each 21-d period. Arterial AA concentrations were reduced by dietary fat treatment and increased by abomasal sodium caseinate infusion. Mammary arteriovenous differences of essential AA tended to increase with both abomasal casein infusion and dietary fat treatments. With ruminal casein infusion treatments, yellow grease supplementation increased the percentage of extraction of blood essential AA into mammary tissue. Mammary blood flow rate dropped 7% on high fat treatments, preventing an increase in uptakes of critical AA to accompany the improved efficiency of milk synthesis, which was evident from a significantly reduced ratio of mammary blood flow to milk volume, resulting in depressed milk protein content.