We have previously identified 14 aminoacyl tRNAs that are specified by yeast mitochondrial DNA (mtDNA). We now report four more amino acids (Arg, Cys, Trp, Thr) that acylate tRNAs which hybridize with mtDNA. Furthermore one of the two mitochondrial tRNAs that we had earlier demonstrated to be directly charged with glutamic acid responds to glutamine but not to glutamic acid codons. Thus Gln-tRNAGln appears to be formed by transamidation of a missense intermediate Glu-tRNAGln. This brings to 19 the number of amino acids which have corresponding tRNAs specified by mtDNA. Only tRNAAsn has not yet been shown to be a mtDNA transcript. We have also mapped the genes for the newly identified mitochondrial tRNAs, as well as several others that were previously identified but unmapped, by hybridization to the mtDNA of a series of petite deletion mutants. We now have ordered 20 mitochondrial tRNA genes (including two methionyl-tRNAs) wtih respect to the antibiotic resistance markers chloramphenicol (CR), erythromycin (ER), paromomycin (PR), and oligomycin I and II (ORI, ORII). Eighteen tRNA genes map between the C and E resistance markers. Only the serinyl-tRNA and glutamyl-tRNA genes are localized near the OI and OII resistance markers.