β-Defensins are cationic peptides with broad-spectrum antimicrobial activity that may play a role in mucosal defenses of several organs. They have been isolated in several species, and in humans, two β-defensins have been identified. Here, we report the identification of two genes encoding β-defensin homologues in the rat. Partial cDNAs were found by searching the expressed-sequence-tag database, and primers were designed to generate full-length mRNA coding sequences. One gene was highly similar to the human β-defensin-1 (HBD-1) gene and mouse β-defensin-1 gene at both the nucleic acid and amino acid levels and was termed rat β-defensin-1 (RBD-1). The other gene, named RBD-2, was homologous to the HBD-2 and bovine tracheal antimicrobial peptide (TAP) genes. The predicted prepropeptides were strongly cationic, were 69 and 63 residues in length for RBD-1 and RBD-2, respectively, and contained the six-cysteine motif characteristic of β-defensins. The β-defensin genes mapped closely on rat chromosome 16 and were closely linked to the α-defensins genes, suggesting that they are part of a gene cluster, similar to the organization reported for humans. Northern blot analysis showed that both RBD-1 and RBD-2 mRNA transcripts were ∼0.5 kb in length; RBD-1 mRNA was abundantly transcribed in the rat kidney, while RBD-2 was prevalent in the lung. Reverse transcription-PCR indicated that RBD-1 and RBD-2 mRNAs were distributed in a variety of other tissues. In the lung, RBD-1 mRNA expression localized to the tracheal epithelium while RBD-2 was expressed in alveolar type II cells. In conclusion, we characterized two novel β-defensin homologues in the rat. The rat may be a useful model to investigate the function and contribution of β-defensins to host defense in the lung, kidney, and other tissues.