Investigations were to determine if bovine mammary tissue could synthesize orotate and to assess the influence of amino acids on production of orotate. Mammary tissue from 6 Holstein cows was obtained immediately after slaughter. Tissue slices were incubated in either a modified Krebs-Ringer bicarbonate buffer, the same medium containing a physiological concentration of amino acids, or a third medium containing a fourfold concentration of the amino acid mixture. In all experiments, tissue slices were preincubated in the physiological amino acid medium for 5, 10, and 15 min (30 total min) with fresh medium changes after each incubation with one of the three treatment media for three consecutive 20-min periods. Tissue slices incubated in the physiological amino acid medium for a total of 90 min synthesized 28.5 microgram orotate/g wet tissue after correction for the initial tissue contribution. Orotate secreted by the tissue slices increased as concentration of amino acids increased. Lactose released into the medium was not affected by amino acid concentration. Bovine mammary tissue can synthesize substantial amounts of orotate, and tissue synthesis of orotate but not lactose increased as amino acid concentration increased.