The effect of transformation by oncogenic Rous sarcoma viruses on the replication of mitochondrial DNA (mtDNA) in chick embryo fibroblasts (CEF) was investigated, extending our previous report of a three- to five-fold increase in the rate of mtDNA replication, which is strictly linked to the expression of the transformed state, is mitochondria-specific, and is not attributable to virus production per se or different growth rates between normal and transformed CEF. In this paper, in vivo pulse-label and pulse-chase analysis shows an increased specific activity in all the replicative and topological forms of transformed cell mtDNA I, labeled within a 10-min pulse, 30-min chase period, reflecting about the same proportion of total label incorporated into D-loop strands (approximately 9S) relative to full-length closed circular forms (approximately 37S) of mtDNA from both cell types. In contrast to the concomitant changes observed in many other systems with elevated DNA synthesis, neither the estimated intramitochondrial pool size of the labeled DNA precursor (dTTP), nor the total level of the mtDNA-replicating enzyme (mt gamma-polymerase) is increased in the transformed cells. Notably, however, in both cell types the mitochondrial dTTP pools relative to the mtDNA complement are significantly larger than whole-cell pools relative to the nuclear DNA complement, confirming recent reports in HeLa cells. The solubilized mt gamma-polymerases from normal and transformed CEF, respectively, are both precipitated by 50% ammonium sulfate, inhibited by N-ethylmaleimide, have similar sedimentation coefficients, and exhibit optimal activity when poly(rA) . d(pT)10 is used as the template-primer. On the other hand, the transformed cell enzyme demonstrates an altered response to thiol compounds, a decreased tendency to aggregate during sedimentation, and is significantly less tightly attached to the mitochondria than the normal cell enzyme. We conclude that, as a result of transformation, an increased fraction of mtDNA molecules replicate at a given time, and that this increased replication rate in vivo is correlated with the expression of several altered endogenous properties, which possibly include a modified intramitochondrial structural attachment of the mt gamma-polymerase in situ. This experimental system may be well suitable for use in the identification of regulatory factors which function during the replication of the mitochondrial genome in vivo.