Mitochondrial proliferation was studied in mature female rats following aortic constriction. Mitochondrial DNA (mtDNA) was assayed by a fluorometric method. The conditions for removal of nuclear DNA were developed and verified by assessment of molecular conformation of DNA. The mtDNA concentration in mitochondria increased 2,4, and 7 days post-operatively by 11, 72 and 117% respectively. Comparison with the rates of accumulation of cytochrome c, b, and aa3 indicates that during the first 24 hours of cardiac enlargement the inner mitochondrial components accumulate faster then mtDNA, but during the six subsequent days the rate of mtDNA increment far outstrips that of the cytochromes. These data indicate that the amount of available mtDNA templates is not the only factor regulating the transcriptional and translational processes in the enlarging myocardium. The analysis of population of replicative intermediates of mtDNA have shown dramatic decrease in the frequency of D-loops in preparations obtained from hypertrophied hearts. This observation indicates that the increase in replicative flux of mtDNA is associated with the removal of a block in the conversion of D-loops to other intermediates.