We have studied the evolution of Gpdh in 18 fruitfly species by sequencing 1,077 nucleotides per species on average. The region sequenced includes four exons coding for 277 amino acids and three variable-length introns. Phylogenies derived by a variety of methods confirm that the nominal genus Zaprionus belongs within the genus Drosophila, whereas Scaptodrosophila and Chymomyza are outside. The rate of GPDH evolution is erratic. The rate of amino acid replacements in a lineage appears to be 1.0 x 10(-10)/site/year when Drosophila species are considered (diverged up to 55 million years ago), but becomes 2.3 x 10(-10) when they are compared to Chymomyza species (divergence around 60 My ago), and 4.6 x 10(-10) when species of those two genera are compared with the medfly Ceratitis capitata (divergence around 100 My ago). In order to account for these observations, the rate of amino acid replacement must have been 15 or more times greater in some lineages and at some times than in others. At the nucleotide level, however, Gpdh evolves in a fairly clockwise fashion.