The binding of misonidazole (MISO) to macromolecules in hypoxic cells is believed to require metabolic reduction. Several factors in the cells' environment, such as pH, glucose, lactate and MISO concentration could affect the capacity of metabolic reduction. Modulation of the binding of MISO by these factors was studied by exposing exponential EMT6/Ro cells to MISO under extremely hypoxic conditions. No binding was observed under aerobic conditions. There was no difference in the binding of 0.02 mM MISO at varying concentrations of glucose from 0.015 mM to 5 mM. Thus, for diagnostic purposes with concentrations of MISO lower than 0.02 mM, little effect of glucose concentration is expected. However, with 5 mM MISO, the binding of MISO increased with increasing glucose concentration (3-fold increase after 2 hours incubation in 5 mM glucose relative to 0.015 mM glucose). At intermediate MISO concentrations (0.1 mM to 5 mM); the higher the MISO concentration, the greater was the increase in binding due to 5 mM glucose. There was no detectable effect of lactate (0, 3 and 10 mM) at pH 7.2 on the binding of MISO either in 0.015 mM or 5 mM glucose. However, a decrease of pH (from 7.2 to 6.5) decreased the binding of MISO in 5 mM glucose but not in 0.015 mM glucose. These data indicated that the binding of MISO is a multi-step process, which involves the concentrations of both glucose (probably via reducing equivalents) and MISO.