The first aim of this study was to compare the hypoxia imaging ability of fluorine-18 fluoroerythronitroimidazole ([18F]FETNIM) with that of fluorine-18 fluoromisonimidazole ([18F]FMISO) in murine tumours of different sizes under two different oxygenation conditions. Secondly, we wanted to assess the biodistribution of the markers in normal tissues under similar conditions. Female CDF1 mice with a C3H mammary carcinoma grown on their backs were used. Tumours were size matched and animals breathed either normal air (21% O(2)) or carbogen gas (95% O(2) + 5% CO(2)). The gassing procedure was begun 5 min before the intravenous injection of either [18F]FETNIM or [18F]FMISO and continued until the mice were sacrificed at 120 min. Blood, tumour, muscle, heart, lung, liver, kidney and fat were removed, counted for radioactivity and weighed. The tumour and muscle were frozen and cut with a cryomicrotome into sections. The spatial distribution of radioactivity from the tissue sections was determined with digital autoradiography. Estimation of the necrotic fraction was made on sections from formalin-fixed tumours. Digital autoradiography showed that the whole tumour-to-muscle radioactivity uptake ratios were significantly higher in normal air-breathing mice than in carbogen-treated mice for both [18F]FETNIM (4.9+/-2.6 vs 1.8+/-0.5; P<0.01) and [18F]FMISO (4.4+/-1.0 vs 1.5+/-0.4; P<0.01). The carbogen treatment had only slight effects on the biodistribution of either marker in normal tissues. The necrotic fraction determined in tumours did not correlate with the tumour volume or with the tumour-to-muscle radioactivity uptake ratio. This study shows that the uptake of both [18F]FETNIM and [18F]FMISO correlates with the oxygenation status in tumours. In addition, our data show no significant difference in the intratumoral uptake between the two markers. However, significantly higher radioactivity uptake values were measured for [18F]FMISO than for [18F]FETNIM in normal tissues.