A simultaneous determination of orotic acid, uric acid, and creatinine in milk is described. Following deproteinization, the sample was analyzed by reversed-phase liquid chromatography, using a highly aqueous cationic ion-pair eluent and photodiode array UV detection. In view of their potential dietary significance, the validated method was applied to survey the influence of species, season, and lactation on their contribution to the nonprotein nitrogen pool in milk. Mature bovine milk contained orotic acid, uric acid, and creatinine in the range of 30-70, 9-24, and 6-12 microg/mL, respectively. Although uric acid and creatinine were present in all milks, orotic acid was essentially absent in nonruminant milks. In contrast to urate and creatinine, expression of orotic acid in bovine milk was strongly dependent on stage of early lactation. The co-existence in mammalian milks of related nucleoside and nucleotide components was also determined.