The nucleotide sequences of genes for small 12 S ribosomal RNA and two transfer RNAs (tRNAPhe and tRNAVal) and their common upstream region in cloned rat mitochondrial DNA were determined. This DNA sequence of 1709 bp extends between the HindII and HindIII cleavage sites in the EcoRI-A and EcoRI-D fragments, respectively. There is no long reading frame for protein synthesis starting from the HindIII site and extending all the way to the HindII site, or vice versa. The heavy strand was found to be the template for these RNA transcripts. The 5' and 3' ends of the 12 S ribosomal RNA gene are contiguous with the tRNAPhe and tRNAVal genes, respectively. The structural features of the two tRNA genes are significantly different from the standard prokaryotic tRNA pattern. Since similar contiguous natures of these genes in human (Crews and Attardi, Cell 19 (1980) 775; Eperon et al., Nature 286 (1980) 460) and mouse (Van Etten et al., Cell 22 (1980) 157) mitochondrial DNA have been reported, comparative studies on the structures of these genes were performed by computer analysis and the characteristic features are discussed.