To gain insight into the role of the general transcription factor,Sp1, in the expression of nuclear genes involved in mitochondrial biogenesis,we investigated Sp1 activation of the adenine nucleotide translocator 2,cytochrome c1, F1-ATPase β subunit, and themitochondria transcription factor (mtTFA) promoters transfected intoDrosophila cell lines. The numbers and organization of GC elementsvary in the four promoters, but the magnitude of activation by coexpressedhuman Sp1 was similar. A feature common to the four promoters is the presenceof multiple, proximal Sp1-activating elements that account for 50% ormore of the transcription activation by Sp1. The distribution and function ofindividual distal Sp1 elements is less defined and appear to be morepromoter-specific. Finally, data from transfected Drosophila cellsprovide the first direct proof for the involvement of Sp1 in the negativeregulation of the ANT2 promoter and as a possible participant in repressionof the β-subunit promoter. The role of Sp1 in both the positive andnegative regulation of OXPHOS promoters is unique.