The general features of the organization of in vivo transcription of the mammalian mitochondrial DNA (mtDNA) were delineated in the 1970s and early 1980s by a detailed analysis of the mapping and metabolic properties of the transcripts of the two strands of this DNA (1, 2), the elucidation of the RNA processing events (3), and the identification of the initiation sites for transcription (4, 5). A significant advance in our understanding of the mechanism and regulation of this process was made possible by the development in the past ten years of in vitro transcription systems. Although the “in organello” transcription system (6) has provided an approach for investigating the role of the extramitochondrial environment and the energetic requirements for transcription, the “open” systems have opened the way for the dissection of the enzymology and the detailed mechanistic aspects of this process. Two such systems have been established. One utilizes semipurified protein components to promote transcription from appropriate mtDNA templates (7), whereas the other makes use of a mitochondrial lysate programmed by exogenous templates (8) and supplemented, when necessary, with purified transcription termination factor (mTERF) (9). The rationale behind the latter approach is to reproduce as closely as possible the in vivo situation to support the various steps of the transcription process, under conditions amenable to external manipulation. In this chapter, we describe in detail the protocols used in our laboratory for the study of transcription initiation and termination in a mitochondrial lysate from HeLa cells.