To provide definitive insight into the complicated roles of the nitric oxide synthase (NOS) enzymes in ischaemia/reperfusion (I/R) injury of skeletal muscle, experiments were undertaken in mice with targeted disruption of the inducible NOS (NOS-2 KO) isoform, compared with the wild-type mouse strain. The degree of I/R injury in the NOS-2 KO mice was attenuated relative to that in the wild-type strain. After 70 min of ischaemia (24 h reperfusion), nitroblue tetrazolium (NBT) staining of skeletal muscle showed significant necrosis (40%) in wild-type mice, whilst in NOS-2 KO mice, ischaemia could be prolonged to 90 min before significant necrosis (38%) was apparent. Specific enzyme activities of the mitochondrial respiratory chain enzymes, measured in skeletal muscle homogenates, suggested that direct inhibition of the enzymes is not causal in the I/R injury. Immunohistological examination of skeletal muscle for NOS-2 showed its induction selectively in mast cells. In vitro experiments using bone marrow-derived mast cells showed that NOS-2 induction was associated with increased degranulation of mast cells. These findings suggest that NO generated by induction of NOS-2 has a deleterious effect in I/R injury of skeletal muscle and that NO exerts its damaging effect through factors released by degranulation of mast cells.