The nuclear gene for a new mitochondrial transcription factor (mtf2) was isolated by transformation of mutant pet-ts3504. It was localized on a 6.4 kb fragment of yeast genomic DNA by subcloning and complementation tests. Sequencing of a 1.7 kb DNA fragment revealed an open reading frame of 1320 bp. A transcript of 1400 nucleotides can be assigned to this region. Gene disruption of this reading frame in a wild-type yeast strain created a stable pet phenotype. Further analysis of this insertion mutation showed that it is allelic to the mutated gene of pet-ts3504. Comparison of the 5' upstream regions of MTF2 and a previously characterized mitochondrial transcription factor (MTF1) revealed common sequence motifs which may be important for coordinated regulation of gene expression.