Purpose:To examine relationships between tumour hypoxia, perfusion and metabolic microenvironment at the microregional level in three different human squamous cell carcinomas (hSCC). Materials and methods:Nude mice bearing FaDu, UT-SCC-15, and UT-SCC-5 hSCC were injected with pimonidazole hypoxia and Hoechst perfusion markers. Bioluminescence imaging was used to determine spatial distribution of glucose and lactate content in serial tumour sections. Metabolite levels were grouped in 10 concentration ranges. Images were co-registered and at each concentration range the proportion of area stained for pimonidazole and Hoechst was determined in 11-13 tumours per tumour line. Results:The spatial distribution of metabolites in pimonidazole hypoxic and Hoechst perfused areas is characterised by pronounced heterogeneity. In all three tumour lines glucose concentration decreased with increasing pimonidazole hypoxic fraction and increased with increasing perfused area at the microregional level. A weak albeit significant positive correlation between lactate concentration and pimonidazole hypoxic fraction was found only in UT-SCC-5. Lactate concentration consistently decreased with increasing perfused area in all three tumour lines. Conclusions:Both glucose consumption and supply may contribute to the microregional glucose levels. Microregional lactate accumulation in tumours may be governed by clearance potential. The extent of microregional hypoxia cannot be predicted from the lactate concentration indicating that both parameters need to be measured independently.