The sequencing of the cloned Locusta migratoria mitochondrial genome has been completed. The sequence is 15,722 bp in length and contains 75.3% A+T, the lowest value in any of the five insect mitochondrial sequences so far determined. The protein coding genes have a similar A+T content (74.1%) but are distinguished by a high cytosine content at the third codon position. The gene content and organization are the same as in Drosophila yakuba except for a rearrangement of the two tRNA genes tRNAlys and tRNAasp. The A+T-rich region has a lower A+T nucleotide content than in other insects, and this is largely due to the presence of two G+C-rich 155-bp repetitive sequences at the 5'end of this section and the beginning of the adjacent small rRNA gene. The sizes of the large and small rRNA genes are 1,314 and 827 bp, respectively, and both sequences can be folded to form secondary structures similar to those previously predicted for Drosophila. The tRNA genes have also been modeled and these show a strong resemblance to the dipteran tRNAs, all anticodons apparently being conserved between the two species. A comparison of the protein coding nucleotide sequences of the locust DNA with the homologous sequences of five other arthropods (Drosophila yakuba, Anopheles quadrimaculatus, Anopheles gambiae, Apis mellifera, and Artemia franciscana) was performed. The amino acid composition of the encoded proteins in Locusta is similar to that of Drosophila, with a Dayhoff distance twice that of the distance between the fruit fly and the mosquitoes. A phylogenetic analysis revealed the locust genes to be more similar to those of the Dipterans than to those of the honeybee at both the nucleotide and amino acid levels. A comparative analysis of tRNA orders, using crustacean mtDNAs as outgroups, supported this. This high level of divergence in the Apis genome has been noted elsewhere and is possibly an effect of directional mutation pressure having resulted in an accelerated pattern of sequence evolution. If the general assumption that the Holometabola are monophyletic holds, then these results emphasize the difficulties of reconstructing phylogenies that include lineages with variable substitution rates and base composition biases. The need to exercise caution in using information about tRNA gene orders in phylogenetic analysis is also illustrated. However, if the honeybee sequence is excluded, the correspondence between the other five arthropod sequences supports the findings of previous studies which have endorsed the use of mtDNA sequences for studies of phylogeny at deep levels of taxonomy when mutation rates are equivalent.