Lysozyme is a 14kDa cationic protein which is bacteriolytic and plays a role in innate immunity. In the gastrointestinal (GI) tract of the human and mouse, lysozyme is synthesised and secreted by specialised granular epithelial Paneth cells which are located in the crypts of the intestinal villi. The aim of this study was to investigate lysozyme expression and synthesis in the chicken (Gallus gallus) avian gastro-intestinal epithelium. Lysozyme was identified in the gut mucosa of five day old birds using RP-HPLC, an antimicrobial assay involving a Salmonella typhimurium phoP mutant and MALDI-TOF mass spectrometry. To identify the intestinal cells synthesising the protein immunohistochemistry was performed on TS sections from the GI tract of 17 day old birds using a 1: 100 dilution of a polyclonal antibody to hen egg white lysozyme, and a secondary antibody conjugated to FITC. Comparable mouse GI sections were stained in parallel. In the mouse fluorescent staining was localised to the base of the intestinal crypts. In contrast staining was detected on the epithelial surfaces of the avian tissues and no staining was observed at the base of the intestinal villi. This suggested that in the avian gut lysozyme was synthesised by intestinal epithelial cells and not by specialised Paneth cells. The GI expression of lysozyme was investigated using reverse transcriptase polymerase chain reaction (RT-PCR) and total RNA isolated from the GI tract of birds aged 4, 17 and 38 days. Three sets of primers were designed to amplify cDNAs encoded by the lysozyme C and chick lysozyme G (Gc) genes, and a third novel lysozyme G gene (Gk). cDNA products of the correct size were generated and confirmed by DNA sequencing. These data show that the avian intestinal epithelial cells synthesise and secrete lysozyme per se and suggest that there are at least three different genes expressing lysozyme in the avian GI tract.