The purpose of this study was to determine how the expression of mitochondrial transcription factor A (Tfam), a protein that governs mitochondrial DNA (mtDNA) transcription and replication, is regulated during a state of reduced organelle content imposed by muscle disuse. We measured Tfam expression at 8 h, 16 h, 24 h, 3 days, or 7 days following denervation and hypothesized that decreases in Tfam expression would precede mitochondrial loss. Muscle mass was lowered by 13% and 38% at 3 and 7 days postdenervation, while cytochrome c oxidase activity fell by 33% and 39% at the same time points. Tfam promoter activation in vivo was reduced by 30-65% between 8 h and 3 days of denervation, while Tfam transcript half-life was increased following 8-24 h of denervation. Protein expression of RNA-binding proteins that promote mRNA degradation (CUG repeat-binding protein and K homology splicing regulator protein) was elevated at 3 and 7 days of denervation. Tfam localization within subsarcolemmal mitochondria was reduced after 3 and 7 days of denervation and was associated with suppression of the cytochrome c oxidase type I transcript at 3 days, indicating that denervation impairs both mitochondrial Tfam import and mtDNA transcription during an early period following denervation. These data suggest that putative signals downregulate Tfam transcription during the earliest stages following denervation but are counteracted by increases in Tfam mRNA stability. Import of Tfam into the mitochondrion seems to be the most critical point of regulation of this protein during the early onset of denervation, an impairment of which is coincident with the loss of mitochondria during muscle disuse.