Bovine alveolar macrophages (BAM) were examined for the expression of β-defensins and to determine whether their expression could be upregulated by bacterial lipopolysaccharide (LPS), as observed with β-defensins expressed in bovine tracheal epithelial cells. Four β-defensins were expressed constitutively in BAM, with bovine neutrophil β-defensin (BNBD)-4 and BNBD-5 being the most predominant. This is the first evidence of β-defensin gene expression in a mature myeloid cell. LPS had no effect on β-defensin expression in BAM, even though tumor necrosis factor alpha (TNF-α) production was induced. Nonbacterial inflammatory particles had little effect on β-defensin gene expression or TNF-α production in BAM. We hypothesize that constitutively expressed β-defensins of alveolar macrophages may have a role in lung host defense.