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The role of the messenger molecule nitric oxide has not been evaluated in fracture healing. NO is synthesized by three kinds of nitric oxide synthase (NOS): inducible NOS (iNOS), endothelial (eNOS), and neuronal (bNOS). We evaluated the role of these enzymes in a rat femur fracture-healing model. There was no messenger RNA (mRNA) expression, immunoreactivity, or enzymatic activity for NOS in unfractured femoral cortex. After fracture, however, mRNA, protein, and enzymatic activity for iNOS were identified in the healing rat femoral fracture callus, with maximum activity on day 15. The mRNA expression for eNOS and bNOS was induced slightly later than for iNOS, consistent with a temporal increase in calcium-dependent NOS activity that gradually increased up to day 30. mRNA expression for the three NOS isoforms also was found in six of six human fracture callus samples. To study the effect of suppression of NO synthesis on fracture healing, an experimental group of rats was fed an NOS inhibitor, l-nitroso-arginine methyl ester (l-NAME), and the control group was fed its inactive enantiomer, d-nitroso-arginine methyl ester (d-NAME). An 18% (p 0.01) decrease in cross-sectional area and a 45% (p 0.05) decrease in failure load were observed in the NOS-inhibited group on day 24 after fracture. Furthermore, the effect of NO supplementation to fracture healing was studied by delivering NO to the fracture site using carboxybutyl chitosan NONOate locally. On day 17 after fracture, there was a 30% (p 0.05) increase in cross-sectional area in the NO-donor group compared with the NOS inhibition group. These results show for the first time that NO is expressed during fracture healing in rats and in humans, that suppression of NOS impairs fracture healing, and that supplementation of NO can reverse the inhibition of healing produced by NOS inhibitors.