The ribonucleotide reductase inhibitor hydroxyurea exhibits potent synergism, even at low, non-cytotoxic concentrations, with the anti-HIV-1 dideoxynucleoside 2',3'-dideoxyinosine, bringing about failure of HIV DNA synthesis and, thus, of HIV replication. To elucidate the incompletely defined role of hydroxyurea in the hydroxyurea/dideoxyinosine interaction and, in particular, to identify the reasons for the unusual selective inhibitory action of the combination on retroviral rather than on cellular DNA synthesis, we prepared specific cDNA probes to determine the effects of low-level hydroxyurea on mammalian cell ribonucleotide reductase M1 and M2 subunit mRNA, while simultaneously quantitating the effects of the drug on cell cycle and on deoxynucleoside triphosphate pools. While dTTP, dCTP, and dGTP pools changed little or even increased in the presence of low-level hydroxyurea, there took place a rapid and specific inhibition of M2-subunit-catalyzed generation of dATP, with consequent slowing of cellular DNA synthesis and prolongation of S phase. However, the latter effect, in turn, resulted in increased M2 subunit mRNA transcription (a process blocked in Go/G1-phase cells, with full-length functional M2 transcripts being generated only during S phase) and, hence, in a return to normal levels of dATP and to a normal rate of cellular DNA synthesis. Because of this self-regulating mechanism, hydroxyurea-induced host-cell toxicity was obviated under conditions where HIV DNA synthesis, a process sensitive to both dATP depletion and the chain-terminating properties of the other inhibitory component of the combination (ddATP derived from dideoxyinosine), was unable to recover.