Conditions for efficient replication in vitro of mitochondrial DNA L strand into H strand products have been established. Gel electrophoresis and hybridization analyses of the products show that neosynthesized H strands are progressively elongated from the D-loop region, and some of them are synthesized as full-length molecules. Evidence for initiation of these H strands de novo is presented. In contrast, there is no detectable L strand synthesis in vitro in this system. This may prove useful for analyzing the distinct molecular mechanisms operating at OH and OL. Use of specific inhibitors indicates that DNA synthesis in the mitochondrial lysate in vitro requires DNA polymerase gamma. These observations support the conclusion that replication in vitro in this system closely resembles the first steps of mitochondrial DNA replication in vivo.