Alterations in mitochondrial activity resulting from defects in mitochondrial DNA (mtDNA) can modulate the biogenesis of mitochondria by mechanisms that are still poorly understood. In order to study mitochondrial biogenesis in cells with impaired mitochondrial activity, we used rho-L929 and rho0143 B cells (partially and totally depleted of mtDNA, respectively), that maintain and even up-regulate mitochondrial population, to characterize the activity of major transcriptional regulators (Sp1, YY1, MEF2, PPARgamma, NRF-1, NRF-2, CREB and PGC-1α) known to control the expression of numerous nuclear genes encoding mitochondrial proteins. Among these regulators, cyclic AMP-responsive element binding protein (CREB) activity was the only one to be increased in mtDNA-depleted cells. CREB activation mediated by a calcium-dependent pathway in these cells also regulates the expression of cytochrome c and the abundance of mitochondrial population as both are decreased in mtDNA-depleted cells that over-express CREB dominant negative mutants. Mitochondrial biogenesis in mtDNA-depleted cells is also dependent on intracellular calcium as its chelation reduces mitochondrial mass. Despite a slight increase in mitochondrial mass in mtDNA-depleted cells, the mitochondrial protein import activity was reduced as shown by a decrease in the import of radiolabeled matrix-targeted recombinant proteins into isolated mitochondria and by the reduced mitochondrial localization of ectopically expressed HA-apo-aequorin targeted to the mitochondria. Decrease in ATP content, in mitochondrial membrane potential as well as reduction in mitochondrial Tim44 abundance could explain the lower mitochondrial protein import in mtDNA-depleted cells. Taken together, these results suggest that mitochondrial biogenesis is stimulated in mtDNA-depleted cells and involves a calcium-CREB signalling pathway but is associated with a reduced mitochondrial import for matrix proteins. © 2005 FEBS.