The aim of this study was to compare a non-invasive 18F-fluoromisonidazole ([18F]FMISO) PET assessment of tumour hypoxia with invasive Eppendorf pO2 measurements in 150-1,500 mm3 C3H mammary carcinomas transplanted on the back of CDF1 mice. The tumour-bearing mice breathed either carbogen gas (95% oxygen, 5% CO2) or normal air during both examinations. Additional autoradiography was performed in separate tumours treated similarly. The PET [18F]FMISO examination significantly discriminated between tumours of carbogen and air-breathing mice. For the pO2 measurements, there was a significantly lower percentage of measurements below 2.5 mmHg for carbogen-treated mice compared with air-breathing mice. However, no direct correlation between the methods was seen. A correlation was found between tumour volume and Eppendorf estimates of tumour hypoxia for the animals breathing normal air, but no correlation was found between the PET endpoint and tumour volume. This may be due to low pO2 measurements obtained in necrotic tissue. Autoradiography confirmed lower [18F]FMISO uptake in tumours of carbogen-breathing animals compared with air-breathing animals, and demonstrated the heterogeneity of the tracer uptake in small compared with larger tumours.