Bovine colostrum and milk samples were collected from two herds over the course of the first month post-partum, pooled for each herd by stage of lactation and total potentially available nucleosides were determined. Sample analysis consisted of parallel enzymatic treatments, phenylboronate clean-up, and liquid chromatography to quantify contributions of nucleosides, monomeric nucleotides, nucleotide adducts, and polymeric nucleotides to the available nucleosides pool. Bovine colostrum contained high levels of nucleosides and monomeric nucleotides, which rapidly decreased as lactation progressed into transitional milk. Mature milk was relatively consistent in nucleoside and monomeric nucleotide concentrations from approximately the tenth day post-partum. Differences in concentrations between summer-milk and winter-milk herds were largely attributable to variability in uridine and monomeric nucleotide concentrations.