Hydroxyurea is an inhibitor of ribonucleotide reductase and is specifically directed at the non-heme iron subunit (which contains the free radical) of this enzyme. Leukemia L1210 cells, grown in the presence of increasing concentrations of hydroxyurea, developed resistance to hydroxyurea. For hydroxyurea, the wild-type L1210 cells required a drug concentration of 85 microM to inhibit cell growth by 50%, and the hydroxyurea-resistant (HU-7-S7) cells required a concentration of approximately 2000 microM. The resistant L1210 cells were cross-resistant to 2,3-dihydro-1H-pyrazolo[2,3-a]imidazole/Desferal. However, these HU-7-S7 cells remained sensitive to 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone and 1-isoquinolylmethylene-N-hydroxy-N'-amino-guanidine tosylate (inhibitors directed at the same subunit as hydroxyurea). The HU-7-S7 cells retained their sensitivity to deoxyadenosine/erythro-9-(2-hydroxy-3-nonyl)adenine and deoxyguanosine/8-amino-guanosine (inhibitors directed at the effector-binding subunit of ribonucleotide reductase). The L1210 cells developed for resistance to hydroxyurea were sensitive to the non-ribonucleotide reductase inhibitors, methotrexate and 1-beta-D-arabinofuranosylcytosine. Ribonucleotide reductase activity was elevated in the HU-7-S7 cells (CDP reductase, 5.5-fold increase; ADP reductase, 13.2-fold increase). The addition of exogenous effector-binding subunit caused much greater stimulation of reductase activities in the extracts from the resistant cells than from the wild-type cells. The reductase activity in cell-free extracts from the resistant cells was inhibited by hydroxyurea, 2,3-dihydro-1H-pyrazolo[2,3-a]imidazole and dATP to the same extent as the activity from the wild-type L1210 cells. These data indicate that resistance to hydroxyurea in these L1210 cells is to some extent related to increased reductase activity. However, the specificity of resistance of these L1210 cells to inhibitors of ribonucleotide reductase depends on the nature of the inhibitor and the subunit at which the inhibitor is directed.