The contents of mitochondrial DNA (mtDNA) and the steady-state amounts of 12 and 16 S mitochondrial rRNAs and the mRNAs for cytochrome c oxidase subunits I and II (COI and COII) were determined in dot hybridization experiments with cloned mtDNA fragments as probes during development from the one-cell to the blastocyst stage. The mtDNA content remained constant during this period at about 2.13 pg or 119,000 mtDNA molecules per embryo, suggesting an absence of mtDNA replication. The amounts of mitochondrial rRNA and the mRNAs for COI and COII varied markedly depending on developmental stage. They remained low between the end of oocyte growth and the late two-cell stage but increased 25-50X during cleavage from two-cell to early blastocyst. In the early blastocyst, the number of mitochondrial mRNA molecules was estimated at 7.9 X 10(6) or about 23% of the total embryo poly(A)+ RNA. These results suggest that the mitochondrial genome is largely inactive in the egg and two-cell embryo but that a high rate of mitochondrial transcription is initiated during cleavage. The activation of the mitochondrial genome coincides with a pronounced structural and functional differentiation of the mitochondria.