Human mitochondrial DNA is transcribed from two distinct, strand-specific promoters located in the displacement loop region of the genome. The transcriptional control sequences identified by deletion mapping and site-directed mutagenesis studies span short regions surrounding the initiation sites and bear no obvious sequence homology to any nuclear or procaryotic promoters. In vitro transcription analyses also revealed several minor initiation sites that are characterized by a pyrimidine-rich region followed by a purine-rich region, a feature that is shared by the two major promoters. In this paper, we report a new class of minor promoters in human mitochondrial DNA. These minor promoters were localized to the same duplex DNA sequences that direct major transcriptional events, but they had transcriptional polarity opposite to that of the major promoters. Furthermore, nucleotide changes that affected the major form of transcription similarly affected transcription in the opposite direction. For one of these minor promoters, a corresponding in vivo RNA species initiating from the same site was identified. These observations indicate that the major transcriptional promoters in human mitochondria can function bidirectionally both in vivo and in vitro.