Recombinant mitochondrial transcription factor 1 (r-mtTF1) was overproduced in Escherichia coli, and purified to near homogeneity by DNA affinity chromatography. Binding affinities of r-mtTF1 to the light strand promoter (LSP) and heavy strand promoter (HSP) of human mitochondrial DNA and a non-promoter fragment derived from pUC vector were quantitatively measured by electrophoretic mobility shift assay. The order of the affinities for these DNAs estimated by competition experiments was LSP > HSP = pUC, which was essentially the same as that of transcriptional activity from each promoter in vitro. Recombinant mtTF1 bound more tightly to supercoiled or relaxed DNA than to linear DNA, moreover to single-stranded DNA with the same affinity for linear DNA. The results suggest that the mechanism of activation of HSP by mtTF1 is different from that of LSP, and that multiple binding of mtTF1 to mitochondrial DNA, in a non-specific manner, may significantly participate in activation of transcription from HSP and replication of mitochondrial DNA.