In pyrimidine biosynthesis the oxidation of dihydroorotate is catalyzed by dihydroorotate dehydrogenase, which is linked to the mitochondrial electron transport chain in vertebrates. In order to elucidate the role of this oxygen-dependent anabolic process in the arrest of cell proliferation under anaerobic culture conditions, the effect of the antimetabolite dihydro-5-azaorotic acid on growth, metabolism and cell cycle distribution of Ehrlich ascites tumor cells was studied and compared with the effect of oxygen-free culture conditions. Experiments with cell homogenates confirmed that the dehydrogenation of dihydroorotate is blocked by the inhibitor. In intact cells 2 mM dihydro-5-azaorotic acid inhibited incorporation of dihydro[6-14C]orotate into nucleic acids, and no further increase in RNA and DNA content was observed in its presence for several hours. The cells remained viable; glycolytic activity was normal; respiration was reduced; growth cessation occurred within a few hours. DNA histograms obtained by flow cytometry revealed an accumulation of cells in the G1 phase, which was also observed with anaerobically cultured cells. Substitution of cultures with pyrimidine nucleosides completely sustained cell proliferation in the presence of the inhibitor but not in an anoxic atmosphere. It is concluded that in the absence of oxygen cell proliferation may be arrested by inhibition of the dihydroorotate dehydrogenase alone. However, additional impairments of cell metabolism must play an important role.