The gene for iso-1-cytochrome c from Saccharomyces cerevisiae was recloned into a pSP65 vector containing an active bacteriophage SP promoter. The iso-1-cytochrome c gene was cloned as an 856-base pair XhoI-HindIII fragment. When the resulting plasmid was digested at the HindIII site 279 bases downstream from the termination codon of the gene and transcribed in vitro using SP6 RNA polymerase, full length transcripts were produced. The SP6 iso-1-cytochrome c mRNA was translated using a rabbit reticulocyte lysate system, and the protein products were analyzed on sodium dodecyl sulfate-polyacrylamide gels. One major band with a molecular weight of 12,000 was detected by autofluorography and coincided with the Coomassie staining band of apocytochrome c from S. cerevisiae. The product was also shown to be identical with that of standard yeast apocytochrome c on an isoelectrofocusing gel. The in vitro synthesized iso-1-apocytochrome c was enzymatically methylated by adding partially purified S-adenosyl-L-methionine:cytochrome c-lysine N-methyltransferase (protein methylase III, EC 2.1.1.59) from S. cerevisiae along with S-adenosyl-L-methionine to the in vitro translation mixtures. The methylation was shown to be inhibited by the addition of the methylase inhibitor S-adenosyl-L-homocysteine or the protein synthesis inhibitor puromycin. The principal type of methylated amino acid in the protein was found to be epsilon-N-trimethyllysine which accounted for 77% of the total. Finally, the methylation of in vitro synthesized iso-1-apocytochrome c was found to increase its import into mitochondria isolated from S. cerevisiae 2-4-fold over unmethylated protein, but not into rat liver mitochondria. This suggests that methylation facilitates the import of apocytochrome c into mitochondria by a specific receptor mechanism.