Mammalian skeletal muscle contains fructose-1,6-bisphosphatase (Fru-1,6-P(2)ase), a key enzyme of glyconeogenesis. We have shown previously that muscle Fru-1,6-P(2)ase is encoded by a gene different from that coding for the liver isoenzyme. Starting with genomic YAC libraries and based on the cDNA sequences of human and mouse muscle Fru-1,6-P(2)ases together with the known gene structures of two mammalian liver fructose-1,6-bisphosphatases, we have PCR-amplified and sequenced all functional parts of the human and mouse muscle fructose-1,6-bisphosphatase genes and determined their chromosomal localization. The human gene (FBP2), localized at chromosome 1p36.1-2, spans about 30 kb, while the mouse gene (Fbp2) at chromosome 13B3-C1 is more compact (about 21 kb). Intron lengths are only poorly conserved between the two genes, while intron number and positions are identical in all hitherto analyzed mammalian fructose-1,6-bisphosphatase isoenzyme genes. Transcriptional start sites were found to be located 97 and 95bp before the start codon in the human gene and 35 bp before the start codon in the mouse homolog. A comparison of the 5'-flanking sequences of the two genes revealed a 56% homology up to human bp -607 before the first transcriptional start point, while upstream of this region we found no similarity. The data presented in this paper provide a basis for further studies of the mechanism of expression regulation and the elucidation of the physiological role of the enzyme.