A resurgence of interest in mitochondrial physiology has recently developed as a result of new experimental data demonstrating that mitochondria function as important participants in a diverse collection of novel intracellular signaling pathways. Cells depleted of mitochondrial DNA, or rho0 cells, lack critical respiratory chain catalytic subunits that are encoded in the mitochondrial genome. Although rho0 cells contain petit mitochondria, they cannot support normal oxidative phosphorylation and must survive and replicate using ATP derived solely from glycolysis. Without a functional electron transport chain, rho0 cells cannot normally regulate redox potential and their mitochondria appear to be incapable of generating reactive oxygen species. Emerging evidence suggests that these signals are important components in a number of mitochondria-initiated signaling pathways. The present article focuses on how rho0 cells have contributed to an understanding of the role that mitochondria play in distinct physiological pathways involved with apoptosis, glucose-induced insulin secretion, and oxygen sensing.