Granulysin (GNLY)/NK-lysin (NKL) is an effector antimicrobial cationic peptide expressed in the cytotoxic and natural killer lymphocytes. We report here cDNA sequence (405 bp) encoding the complete functional domain of buffalo-lysin (bu-lysin), and its expression profile in the various tissues. The nucleotide sequence of bu-lysin exhibited >85% identity with the bovine lysin. Comparison of the deduced amino acid sequence of bu-lysin with those of GNLY/NKL of different species revealed the conservation of six cysteine (Cys) residues and five alpha helices. Unlike the homologues in other species, bu-lysin composed of 11 positively charged Lys residues as in equine. The expression of bu-lysin mRNA in the in vitro cultured lymphocytes was inducible and increased markedly ( p < 0.05) in a dose dependant manner when incubated with Concanavalin A (ConA). The expression of bu-lysin mRNA in the different tissues was variable: comparatively higher in the spleen and lymph node, moderate in the uterine endometrium and low in the liver and kidney. These results indicate the existence and active expression of GNLY/NKL homologue in water buffalo having a significant influence in immune response.