The study was designed to investigate the binding of [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluoro-2-deoxy-D-glucose ([18F]FDG) in a C3H mouse mammary carcinoma. Non-anaesthetized tumour-bearing animals breathing either normal air or carbogen (to reduce tumour hypoxia) were examined by PET after tracer injection. Tumours were identified by radioactive labelling and methods of defining regions of interest (ROI) in the tumours were investigated. Reference tissue was selected elsewhere in the mice and the ratio between mean radioactivity in tumour and reference tissue was compared. The results showed a correlation between the methods of identifying ROIs and a significantly lower tumour to reference tissue ratio for carbogen-treated mice compared with controls when using [18F]FMISO. Only one of the methods showed a significant difference in the tumour labelling between treatment groups using [18F]FDG. The study supports the contention that [18F]FMISO may be able to identify hypoxia in tumours, whereas a similar role for [18F]FDG is more doubtful.