The first cartilaginous fish iNOS gene has been cloned in the small spotted catshark, Scyliorhinus canicula. The cDNA was 4568 bp long, with a 3375 bp open reading frame encoding a protein of 1125 amino acids and a predicted molecular mass of 127.8 kDa. The catshark translation had 77% amino acid similarity with chicken iNOS and 70-73% similarity with known teleost i NOS molecules. The various co-factor binding sites were well conserved, with the calmodulin site hydrophobicity profile noticeable more similar to tetrapod molecules than teleost molecules. The catshark iNOS transcript was not typically expressed constitutively, with the exception of the gills. Clear induction of the gene was seen in splenocytes after exposure to Vibrio anguillarum in vivo, and after stimulation with LPS in vitro. iNOS message was first seen 2 h after stimulation, and was still apparent 24 h post-stimulation, the last timing studies. Poly I:C was also able to induce iNOS transcript expression in splenocytes, albeit at a later timing (i.e.24 h). Such findings suggest a role for this molecule in the non-specific defences of cartilaginous fish as seen in other vertebrate groups.