JB3-B is a Chinese hamster ovary cell mutant previously shown to be temperature-sensitive for DNA replication (J.J. Dermody, B.E. Wojcik, H. Du, and H.L. Ozer, Mol. Cell. Biol. 6, 4594-4601 (1986)). Because preliminary evidence suggested that the defective function involved DNA precursor biosynthesis, we investigated the biochemical basis responsible for this mutation. Measurement of deoxyribonucleoside triphosphate (dNTP) pools as a function of time after shift of cultures from 33℃ to 39℃ revealed that all four dNTP pools declined at similar rates in extracts prepared either from whole cells or from rapidly isolated nuclei. Ribonucleoside triphosphate pools were unaffected by a temperature shiftup, ruling out the possibility that the mutation affects nucleoside diphosphokinase. However, ribonucleotide reductase activity, as measured in extracts, declined after cell cultures underwent a temperature shiftup, in parallel with the decline in dNTP pool sizes. Moreover, the enzyme activity in a cell extract was thermolabile in vitro, consistent with the model that the JB3-B mutation affects the structural gene for one of the ribonucleotide reductase subunits. However, the possibility of an indirect effect on ribonucleotide reductase activity in JB3-B has not been excluded since ribonucleotide reductase was temperature-stable in the cells transfected with human sequences other than those encoding the subunits of this enzyme.