Fusions between the TRMI gene of Saccharomyces cerevisiae and COXIV or DHFR were made to examine the mitochondrial targeting signals of N2,N2-dimethylguanosine-specific tRNA methyltransferase [tRNA (m2G)dimethyltransferase]. This enzyme is responsible for the modification of both mitochondrial and cytoplasmic tRNAs. We have previously shown that two forms of the enzyme are translated from two in-frame ATGs in this gene, that they differ by a 16-amino-acid amino-terminal extension, and that both the long and short forms are imported into mitochondria. Results of studies to test the ability of various TRMI sequences to serve as surrogate mitochondrial targeting signals for passenger protein import in vitro and in vivo showed that the most efficient signal derived from tRNA (m2G)dimethyltransferase included a combination of sequences from both the amino-terminal extension and the amino terminus of the shorter form of the enzyme. The amino-terminal extension itself did not serve as an independent mitochondrial targeting signal, whereas the amino terminus of the shorter form of tRNA (m2G)dimethyltransferase did function in this regard, albeit inefficiently. We analyzed the first 48 amino acids of tRNA (m2G)dimethyltransferase for elements of primary and secondary structure shared with other known mitochondrial targeting signals. The results lead us to propose that the most efficient signal spans the area around the second ATG of TRMI and is consistent with the idea that there is a mitochondrial targeting signal present at the amino terminus of the shorter form of the enzyme and that the amino-terminal extension augments this signal by extending it to form a larger, more efficient mitochondrial targeting signal.The TRMI gene of Saccharomyces cerevisiae codes for an enzyme that catalyzes the modification of a specific guanosine to N2,N2-dimethylguanosine in both cytoplasmic and mitochondrial tRNAs (17). In previous reports, we described the isolation and characterization of the TRMJ gene from S. cerevisiae (14,15 (24), FUMI (38), LEU4 (3), and VASI (6) loci of S. cerevisiae each code for two enzymes that differ by an amino-terminal extension, and in each case it has been shown that the amino-terminal extension functions to direct the enzyme to an intra-or extracellular location different from that of the enzyme lacking the aminoterminal extension.Since the amino-terminal extension of tRNA (m2G)