The complete mitochondrial genome sequences were determined for two species of human hookworms, Ancylostoma duodenale (13,721 bp) and Necator americanus (13,604 bp). The circular hookworm genomes are amongst the smallest reported to date for any metazoan organism. Their relatively small size relates mainly to a reduced length in the AT-rich region. Both hookworm genomes encode 12 protein, two ribosomal RNA and 22 transfer RNA genes, but lack the ATP synthetase subunit 8 gene, which is consistent with three other species of Secernentea studied to date. All genes are transcribed in the same direction and have a nucleotide composition high in A and T, but low in G and C. The AT bias had a significant effect on both the codon usage pattern and amino acid composition of proteins. For both hookworm species, genes were arranged in the same order as for Caenorhabditis elegans, except for the presence of a non-coding region between genes nad3 and nad5. In A. duodenale, this non-coding region is predicted to form a stem-and-loop structure which is not present in N. americanus. The mitochondrial genome structure for both hookworms differs from Ascaris suum only in the location of the AT-rich region, whereas there are substantial differences when compared with Onchocerca volvulus, including four gene or gene-block translocations and the positions of some transfer RNA genes and the AT-rich region. Based on genome organisation and amino acid sequence identity, A. duodenale and N. americanus were more closely related to C. elegans than to A. suum or O. volvulus (all secernentean nematodes), consistent with a previous phylogenetic study using ribosomal DNA sequence data. Determination of the complete mitochondrial genome sequences for two human hookworms (the first members of the order Strongylida ever sequenced) provides a foundation for studying the systematics, population genetics and ecology of these and other nematodes of socio-economic importance.