We describe our preliminary studies on the development of methods to measure hypoxia in standard paraffin sections of human tumors. Three parameters were investigated. First, image analysis of tumor vascularity yielded the parameter diffusion limited fraction (DLF), which is the amount of tumor tissue greater than a fixed distance from the nearest blood vessel. Secondly, the amount of tumor tissue stained with antibodies against bound reduced products of the bioreductive marker pimonidazole was assessed. Finally, the fraction of blood vessels showing no surrounding tumor tissue labeled with lUdR, a cell kinetic marker, was measured. DLF and pimonidazole monitor primarily chronic hypoxia, while it is hypothesized that the IUdR-negative fraction monitors acute hypoxia. Feasibility was demonstrated in a series of 10 esophageal and 10 rectal tumors (no drug administration), 10 cervix tumors (pimonidazole) and 14 head and neck tumors (pimonidazole and lUdR). Significant differences between tumors were found for all parameters. DLF correlated significantly with the pimonidazole fraction when all images of all tumors were included, although mean values per tumor showed no correlation. The IUdR-negative fraction did not correlate with either of the other two parameters. We conclude that it is feasible to measure hypoxia-related, and possibly perfusion-related, parameters on paraffin sections for predictive purposes, although each method needs further validation. Each parameter will be correlated with outcome in a larger study on head and neck tumors treated with surgery with or without postoperative radiotherapy.