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We have shown previously that nitric oxide (NO) has regulatory effects on fracture healing. Our aim here was to investigate the temporal expression patterns of the three NO synthase (NOS) isoforms that are responsible for the generation of NO by semiquantitative competitive polymerase chain reaction (PCR) and immunoblot analysis after femoral fractures in rats. We found that 4 days after fracture, there were increases in the levels of messenger RNA (mRNA) for all three NOS isoforms, with peaks for the inducible NOS (iNOS; 35-fold increase, p < 0.05) at day 4, the endothelial NOS (eNOS; 5-fold increase, p < 0.05) at day 7, and the neuronal NOS (bNOS; 16-fold increase, p < 0.05) at day 21. At a protein level, the time course expression of NOS isoforms was consistent with the results of those at the mRNA level. In addition, we have previously reported a 2.5-fold increase in NOS activity detected by [3H]arginine to [3H]citrulline conversion at day 15 compared with that at day 4 after fracture. The findings that the expression of NOS isoforms during fracture healing is type specific and time dependent are important and may have clinical applications in the regulation of bone repair by NOS inhibitors or stimulators at different stages after injury.