One variant, aphhs-3 was previously isolated based on a hypersensitivity to nontoxic concentrations of aphidicolin, a specific inhibitor of DNA polymerases-alpha and delta. This variant was found to be more sensitive to temperatures above 35 degrees C and to 10 microM of 3'-azido-3'-deoxythymidine (zidovudine, azidothymidine, or AZT) than the parental 743x cells. DNA polymerase activities in the cell extract or in the partially purified fraction by DEAE-cellulose (DE52) anion exchange column from aphhs-3 were active at 40 degrees C. No significant differences in deoxynucleoside triphosphate pools were observed at 34 degrees C for both the parental 743x and aphhs-3 cells. Revertants were isolated at 39 degrees C: six revertants (aphhs-3-tr1 through aphhs-3-tr6) were obtained without aphidicolin; one revertant aphhs-3-tar (the tar clone) was selected in aphidicolin (0.12 microM). The hypersensitivity to aphidicolin (Aphhs) and AZT (AZThs) was cosegregated in the revertant aphhs-3-tr5 (the tr5 clone), while the tar clone was not AZThs. There was a similar increase in the specific activity of 3H-labeled DNA in all cell lines after additions of [3H]AZT or [3H]thymidine. Additions of purine or pyrimidine arabinosides (araT, araC, and araA) to all cell lines resulted in a similar cytotoxicity, suggesting the anabolism of dTTP was not defective in the tr5 clone. The spontaneous mutation rate at the hypoxanthine-guanine phosphoryltransferase locus using replating techniques and 6-thioguanine resistance selection was less than or equal to 5 x 10(-7), 2.2 x 10(-6), or 1.3 x 10(-6) per generation for the tr5, 743x, or tar cell lines, respectively. Most importantly, DNA polymerase activities in the cell extract of the revertant tr5 clone were inhibited by 0.5 microM AZTTP. In contrast, no inhibition was observed in those of the parental 743x and revertant tar cells. The cosegregation of both Aphhs and AZThs in the tr5 revertant suggests that these two phenotypes may be a result of the same mutational event.