We report the biochemical and functional properties of a novel bovine beta-defensin (bBD-1). Cloned from bovine mammary papillary duct epithelia, the bBD-1 cDNA predicts a 69 amino acid propeptide that is much more similar to human beta-defensin-1 (hBD-1) than to other bovine defensins. The bBD-1 gene contains two exons and one 8.5 kb intron. Using RT-PCR, we detected the bBD-1 transcript in the teat mucosa, kidney, vagina, ovary, oviduct, and colon. A synthetic bBD-1 peptide demonstrates potent antibacterial activity against Escherichia coli. The widespread expression of bBD-1 mRNA indicates that bBD-1 may play an important role in the bovine host defense against infections.