We showed that early chronic hypoxia (CH) lasting 1-3 days induces vasodilatation and increased vascular permeability in skeletal muscle of rats (Walsh & Marshall, 2000). By 14 days there is increased arteriolar branching (Smith & Marshall, 1999), and by 3 weeks capillary angiogenesis (Deveci et al. 2001). We have also shown increases in mRNA for inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) in Tibialis Anterior (TA), Soleus (SOL) and Spinotrapezius (SP) muscles in rats exposed to CH from 2 h to 14 days, but no change in endothelial (e)NOS mRNA (Glen et al. 2004). We have now investigated the changes in protein expression and localization of iNOS, VEGF and eNOS. Experiments were performed on normoxic (N) rats and on rats exposed to 12% O₂ (CH) for between 2 h and 14 days (n=6 in each case). Under anaesthesia (Sagatal 60 mg kg^-1) muscles were removed and frozen in liquid N₂-cooled 2-methylbutane. All animals were killed by anaesthetic overdose. Western blots were performed to measure protein expression of iNOS, VEGF and eNOS in TA, SOL and SP muscles. Immunohistochemistry with fluorescent tags was performed on serial sections of SOL and TA from N rats, and rats exposed to 6 h and 1 day CH, to localize these proteins and to detect HIF-1α, which is known to increase the expression of VEGF and iNOS. Alternate sections were stained for muscle fibre types using myosin ATPase and succinate dehydrogenase techniques and with lectin to identify capillaries. No iNOS protein was detected in any muscles, by Western blot or immunohistochemistry. VEGF and eNOS protein was detected in all muscles. However, densitometry analysis showed increases in VEGF protein from 1 day CH and were maintained to 14 day CH, whereas eNOS protein did not change with CH. Immunohistochemistry indicated eNOS in capillary endothelium of muscles in all rats. VEGF was also present in capillaries and is associated with the sarcolemma of glycolytic fibres at 1 day CH. HIF-1α was diffusely present in muscles of N rats, but appeared to be concentrated in the nuclei of muscle fibres at 1 day CH. We suggest that the increase in VEGF protein seen in muscles in the first few days of CH is consistent with the increase in vascular permeability and onset of angiogenesis (see above) and may be triggered by HIF-1α.