Abstract: The absence of mtDNA in rho(0) yeast cells affects both respiration and mitochondrial-nuclear communication (e.g., retrograde regulation, intergenomic signaling, or pleiotropic drug resistance). Previously, it has been reported that some rho(0) strains have increased replicative lifespans, attributable to the lack of respiration and retrograde regulation. Here, we have been able to confirm that rho(0) cells exhibit increased replicative lifespans but have found that this is not associated with the lack of respiration or reduced oxidative stress but instead, is related to the lack of mtDNA per se in rho(0) cells. Also, we find no correlation between the strength of retrograde regulation and lifespan. Furthermore, we find that pdr3(-) or rtg2(-) mutations are not responsible for lifespan extension in rho(0) cells, ruling out a specific role for PDR3-pleiotropic drug resistance or RGT2-retrograde regulation pathways in the extended lifespans of rho(0) cells. Surprisingly, Rtg3p, which acts downstream of Rtg2p, is required for lifespan increase in rho(0) cells. Together, these findings indicate that the loss of mtDNA per se and not the lack of respiration lead to extended longevity in rho(0) cells. They also suggest that Rtg3p, acting independently of retrograde regulation, mediates this effect, possibly via intergenomic signaling.