The chronological lifespan of eukaryotic organisms is extended by the mutational inactivation of conserved growth-signaling pathways that regulate progression into and through the cell cycle. Here we show that in the budding yeast S. cerevisiae, these and other lifespan-extending conditions, including caloric restriction and osmotic stress, increase the efficiency with which nutrient-depleted cells establish or maintain a cell cycle arrest in G1. Proteins required for efficient G1 arrest and longevity when nutrients are limiting include the DNA replication stress response proteins Mec1 and Rad53. Ectopic expression of CLN3 encoding a G1 cyclin downregulated during nutrient depletion increases the frequency with which nutrient depleted cells arrest growth in S phase instead of G1. Ectopic expression of CLN3 also shortens chronological lifespan in concert with age-dependent increases in genome instability and apoptosis. These findings indicate that replication stress is an important determinant of chronological lifespan in budding yeast. Protection from replication stress by growth-inhibitory effects of caloric restriction, osmotic and other stresses may contribute to hormesis effects on lifespan. Replication stress also likely impacts the longevity of higher eukaryotes, including humans. © 2007 Weinberger et al.