Peptide sequences obtained from cyanogen bromide fragments of bovine lactoperoxidase (bLPO) were used to design oligonucleotide probes for library screening. These probes were used to screen a cDNA library constructed from bovine mammary tissue. Three overlapping clones were obtained, the longest of which (T3) contained a reading frame of 712 amino acid residues. The encoded amino acid sequence was homologous to those recently reported for myelo-, thyro-, and eosinophil peroxidases. Two possible amino termini of the mature enzyme were identified, and the predicted mature protein matched previous molecular weight estimates of 78,500. Of eight bovine tissues tested, transcription of T3 sequences were detected in mammary tissue only. Using the bLPO cDNA as a probe, a single hybridizing clone was found in a human mammary gland cDNA library. This clone (M1) encoded the carboxy-terminal 324 residues of a peroxidase distinct from the other three known human peroxidases, and was closely related to bLPO. This result confirms the presence of at least one distinct lactoperoxidase in humans.