The β-defensins are a family of peptides with antimicrobial activity which provide a mechanism of host defence against infectious agents. They are expressed in vertebrate neutrophils and epithelial cells, and their structures are typified by six cysteine amino acids which form three di-sulphide bonds. BLASTp searches of the chicken EST intestinal database identified a clone ChEST203o22 whose predicted amino acid sequence showed homology to that of the bovine β-defensin 9 antimicrobial peptide. Using the BLAST (tblastn) nucleic acid alignment programme chicken (c) β-defensin 9 was identified on chromosome 3 of the draft Gallus gallus genome. The gene comprises three exons and two introns. Analyses of the encoded primary structure of the c β-defensin 9 peptide indicated a putative signal sequence suggesting that the peptide was secreted. Typical of cationic anti-microbial peptides, the peptide was composed of a high percentage of positively charged and hydrophobic amino acids and within this region six conserved cysteine residues were identified. RT-PCR analyses using primers designed to cβ-defensin 9 and sequencing of the cDNA products, revealed the expression of two cDNA fragments (300 and 900bp) in the small intestine, liver, ovary and gizzard tissues of 5-day old chicks. In contrast only the 300 bp cDNA was detected in RNA isolated from the pancreas and white blood cells. These data suggest that cβ-defensin 9 gene expression involves spliced transcripts and is subject to tissue specific regulation. To determine whether the expression of cβ-defensin 9 in the small intestine was constitutive or inducible in response to Salmonella infection, groups of five day-old chickens (n=3 or 4) were gavaged with either S.enteriditis, S.typhimurium SL1344 or PBS. The birds were sacrified after 4 days of infection and the small intestinal RNAs analysed for cβ-defensin expression using semi-quantitative RT-PCR. No change was detected in the expression of the 900bp cDNA fragment in the infected birds compared to controls suggesting that its expression was constitutive. These data provide evidence for a novel cationic antimicrobial peptide which is part of the avian innate host defence system.