The SRL4 (YPL033C) gene was initially identified by the screening of Saccharomyces cerevisiae genes that play a role in DNA metabolism and/or genome stability using the SOS system of Escherichia coli. In this study, we found that the srl4Δ; mutant cells were resistant to the chemicals that inhibit nucleotide metabolism and evidenced higher dNTP levels than were observed in the wild-type cells in the presence of hydroxyurea. The mutant cells also showed a significantly faster growth rate and higher dNTP levels at low temperature (16 oC) than were observed in the wild-type cells, whereas we detected no differences in the growth rate at 30oC. Furthermore, srl4Δ was shown to suppress the lethality of mutations of the essential S phase checkpoint genes, RAD53 and LCD1. These results indicate that SRL4 may be involved in the regulation of dNTP production by its function as a negative regulator of ribonucleotide reductase.