We have used HeLa cells without mitochondrial DNA (ρ0-cells) and transient ρ0-phenocopies, obtained from wild-type cells by short-term treatment with ethidium bromide, to analyze how the absence of a functional mitochondrial respiratory chain slows down proliferation. We ruled out an energetic problem (ATP/ADP content) as well as defective synthesis of pyrimidine, iron-sulfur clusters or heme as important causes for the proliferative defect. Flow cytometric analysis revealed that reactive oxygen species were reduced in ρ0-cells and in ρ0-phenocopies, and that, quite unusually, all stages of the cell cycle were slowed down. Specific quenching of mitochondrial ROS with the ubiquinone analog MitoQ also resulted in slower growth. Some important cell-cycle regulators were reduced in ρ0-cells: cyclin D3, cdk6, p18INK4C, p27KIP1, and p21CIP1/WAF1. In the ρ0-phenocopies, the expression pattern did not fully duplicate the complex response observed in ρ0-cells, and mainly p21CIP1/WAF1 was downregulated. Activities of the growth regulatory PKB/Akt and MAPK/ERK-signaling pathways did not correlate with proliferation rates of ρ0-cells and ρ0-phenocopies. Our study demonstrates that loss of a functional mitochondrial electron transport chain inhibits cell-cycle progression, and we postulate that this occurs through the decreased concentration of reactive oxygen species, leading to downregulation of p21CIP1/WAF1. J. Cell. Physiol. 209: 103–112, 2006. © 2006 Wiley-Liss, Inc.