A comparison of the amino acid sequences of the liver and muscle fructose-1,6-bisphosphatase (FbPase) isoforms in primates and rodents suggested an ancient duplication event leading to the corresponding genes. We investigated the presence of both genes in the rabbit (order lagomorphs) and in species belonging to further distantly related metazoan taxa. By an analysis of the available complete genomes and proteomes of the nematode Caenorhabditis elegans and of Drosophila melanogaster only one sequence homologous to known FbPases was found in each species. The corresponding mRNAs were characterized by cDNA sequencing. We then carried out reverse transcription-polymerase chain reactions to amplify central fragments of the FbPase cDNAs from liver and muscle of Gallus gallus, Xenopus laevis, and Esox lucius, respectively. Their sequencing revealed that (i) the livers of chicken, frog, and fish contain mRNAs which are closely related to mammalian liver FbPase mRNAs, (ii) chicken muscle contains an mRNA which is most homologous to mammalian muscle FbPase mRNAs, (iii) frog muscle contains both a liver-type and a muscle-type FbPase mRNA, while (iv) in fish muscle no FbPase mRNA could be detected by our approach despite the doubtless presence of the enzyme in this organ. An alignment of the partial amino acid sequences of the different FbPases showed that the residues that are thought to be in contact with the substrate, fructose-2,6-bisphosphate, and Mg(2+) are totally conserved, while some amino acids having contact with adenosine monophosphate were found to vary among several species. The question of what might be the advantage of having more than one gene coding for FbPase per haploid genome is discussed.