The flavoenzyme thioredoxin reductase (TR) is an important enzyme for many aspects of cellular function. The antitumor quinones diaziquone and doxorubicin have been shown to produce a time- and concentration-dependent inhibition of TR when incubated for up to 24 hr with intact A204 human rhabdomyosarcoma cells. There was a positive correlation between the inhibition of TR and the inhibition of cell colony formation measured 7 days later for diaziquone (r = 0.84, P less than 0.01), and for doxorubicin (r = 0.87, P less than 0.01). 2,6-Dichloroindophenol, which in previous studies was shown to be a good inhibitor of TR in vitro, was a poor inhibitor of TR in intact A204 cells and there was no significant correlation with inhibition of colony formation. The activity of ribonucleotide reductase, which catalyzes the first unique step of DNA synthesis and which obtains its reducing equivalents from TR through thioredoxin, was decreased in diaziquone- and doxorubicin treated A204 cells. We suggest that the inhibition of TR by some antitumor quinones leading to a decreased activity of TR and, consequently, a decreased activity of thioredoxin-dependent enzymes including ribonucleotide reductase may contribute to the growth inhibitory activity of these quinones.