Using oligonucleotide primers based on mammalian nitric oxide synthases (NOS), expression of an inducible NOS (iNOS) gene was detected in head kidney and gill tissue of bacterially-challenged rainbow trout. Three overlapping fragments were amplified by RT-PCR and used to construct a contiguous sequence of 1410 bp, with high nucleotide homology to iNOS in birds (61%) and mammals (57–59%). The nucleotide sequence translated in one reading frame to produce a partial peptide containing 470 amino acids, with 69–71% amino acid homology with mammalian iNOS, 81% homology with chicken iNOS and 85% homology with a partial (492bp) goldfish iNOS sequence. In vitro stimulation of head kidney macrophages with LPS also induced expression of the trout iNOS RNA, with optimal expression seen using 20–50 μg/ml LPS at 2 h to 6 h post-stimulation. The evolutionary and functional significance of the trout iNOS sequence are discussed.