The relationship between dNTP levels and DNA synthesis was investigated using alpha factor-synchronized yeast treated with the ribonucleotide reductase inhibitor hydroxyurea (HU). Although HU blocked DNA synthesis and prevented the dNTP pool expansion that normally occurs at G(1)/S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G(1) levels. When dbf4 yeast that are ts for replication initiation were allowed to preaccumulate dNTPs at 37 degreesC before being released to 25 degreesC in the presence of HU, they synthesized 0.3 genome equivalents of DNA and then arrested as dNTPs approached sub-G(1) levels. Accumulation of dNTPs at G(1)/S was not a prerequisite for replication initiation, since dbf4 cells incubated in HU at 25 degreesC were able to replicate when subsequently switched to 37 degreesC in the absence of HU. The replication arrest mechanism was not dependent on the Mec1/ Rad53 pathway, since checkpoint-deficient rad53 cells also failed to exhaust basal dNTPs when incubated in HU. The persistence of basal dNTP levels in HU-arrested cells and partial bypass of the arrest in cells that had preaccumulated dNTPs suggest that cells have a mechanism for arresting DNA chain elongation when dNTP levels are not maintained above a critical threshold.