The transcriptional co-activator PGC-1alpha and the NAD(+)-dependent deacetylase SIRT1 are considered important inducers of mitochondrial biogenesis because in the nucleus they regulate transcription of nucleus-encoded mitochondrial genes. We demonstrate that PGC-1alpha and SIRT1 are also present inside mitochondria and are in close proximity to mtDNA. They interact with mitochondrial transcription factor A (TFAM) as assessed by confocal microscopy analysis and by blue native-PAGE. Nucleoid purification allowed us to identify SIRT1 and PGC-1alpha as proteins associated with native and cross-linked nucleoids, respectively. After mtDNA immunoprecipitation analysis, carried out on mitochondrial extracts, we found that PGC-1alpha is present on the same D-loop region recognized by TFAM. Finally, by oligonucleotide pulldown assay, we found PGC-1alpha and SIRT1 associated with the TFAM consensus sequence (human mitochondrial transcription factor-binding site H). The results obtained suggest that in mitochondria PGC-1alpha and SIRT1 may function as their nuclear counterparts and represent the genuine factors mediating the cross-talk between nuclear and mitochondrial genome. Finally, this work adds new knowledge on the function of SIRT1 and PGC-1alpha and highlights the direct involvement of such proteins in regulation of mitochondrial biogenesis.