In the in vitro mitochondrial (mt) transcription initiation system with mt RNA polymerase fraction and mt lysate, the transcription initiation products were shown to be synthesized bidirectionally from the only H-strand-promoter (HSP)/L-strand-promoter region (LSP) of the mitochondrial D-loop genome segment. These transcription products ranged between > 100 and > 800 bp with the purified mitochondrial RNA polymerase fraction, but were larger (> 2030-4000 bp) in size with the mitochondrial lysate in both human and mouse. In this brief report, an in vitro reconstituted mitochondrial transcription system purified by affinity chromatography (heparin-Sepharose) from mouse hypotetraploid letter Ehrlich ascites tumor cell mitochondria was shown to initiate transcription bidirectionally from the mitochondrial D-loop region (HSP/LSP), as evidenced by in vitro generated transcription products. The in vitro generated transcription products were separated by sequencing gel. But this in vitro reconstituted transcription system was not studied beyond the D-loop region. A 3D model of the enzyme RNA polymerase was docked with both ATP and CTP. (c) 2007 Elsevier Inc. All rights reserved.