Technetium-99m HL91 (HL91) is a potential agent for imaging hypoxic tissue in vivo. To elucidate the relationship between hypoxia and blood flow in a tumour, dual-tracer autoradiography with HL91 and carbon-14 iodoantipyrine (IAP) was performed in a tumour-bearing rat model. The distribution of each tracer was analysed visually and semiquantitatively. In the tumours with central necrotic areas, HL91 uptake was marked around the necrotic areas whereas IAP uptake was marked at the periphery of the tumours, around the areas of marked HL91 uptake. Normalized HL91 uptake (%HL91) was highest in the low normalized IAP uptake (%IAP) fraction in the non-necrotic areas. There was a weak negative correlation between %HL91 and %IAP in the non-necrotic areas (r = -0.322, P<0.0001). In tumours with few or no necrotic areas, HL91 uptake was heterogeneous throughout the tumours, while IAP uptake predominantly occurred at the periphery of the tumours. %HL91 was higher in the inner two-thirds of the tumour than in the outer third. There was again a weak negative correlation between %HL91 and %IAP (r = -0.354, P<0.0001). This study confirmed that high HL91 uptake is related to low blood flow. The marked HL91 uptake around the necrotic region suggests the presence of chronic hypoxia in a tumour.