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We investigated the temporal expressions of the three nitric oxide synthase (NOS) isoforms by semi-quantitative polymerase chain reaction (PCR) assays and by immunoblot analysis, following Achilles tendon transection in rats. Four days after injury, there were increases in the steady-state levels of mRNA for all three NOS isoforms, with peaks for the inducible isoform (iNOS) (23-fold increase) at day 4, the endothelial isoform (eNOS) (24-fold increase) at day 7 and the neuronal isoform (bNOS) (seven-fold increase) at day 21. The temporal expression of NOS isoforms at a protein level was consistent with the results at the mRNA level. We have previously shown a five-fold increase in the NOS activity, as detected by 3H-arginine to 3H-citrulline conversion, at day 7 postinjury. These findings indicate that all three NOS isoforms are expressed during tendon healing with differential expression patterns during the various phases of tendon healing. These findings may prove clinically relevant with respect to strategies for regulating tendon healing.