During retroviral DNA synthesis reverse transcriptase frequently performs nonrequired template switches that can lead to genetic rearrangements or recombination. It has been postulated that template switching occurs after pauses in the action of reverse transcriptase. Hence factors which affect pausing, such as polymerization rate, may affect the frequency of template switching. To address the hypothesis that increasing the time required to complete reverse transcription increases the frequency of template switching, we established conditions that lengthened the time required to complete a single round of intracellular Moloney murine leukemia virus reverse transcription approximately threefold. Under these conditions, which resulted from intracellular nucleotide pool imbalances generated with hydroxyurea, we examined template switching frequency using a lacZ-based tandem repeat deletion assay. We observed that the frequency of deletion during reverse transcription in hydroxyurea-treated cells was approximately threefold higher than that in untreated control cells. These findings suggest that rates of retroviral recombination may vary when the intracellular environment is altered.