Only one gene coding for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12), a key enzyme in the control of glycolysis, is known to be functional in man, mouse, rat and chicken. The gene has been localized to chromosome 12 in human and chromosome 6 in mouse. Only a single mRNA species has been found in chicken and rat. However, analysis of genomic DNA blots of various species with a cloned GAPDH cDNA probe has revealed large differences in the level of reiteration, ranging from one to over 200 copies. On this basis, we have grouped these organisms into three classes according to the number of GAPDH-related sequences they contain; one class with a unique representation (chicken), another class of relatively low reiteration (10-30 copies in man, hare, guinea-pig and hamster) and a third class of high reiteration (greater than 200 copies in mouse and rat). The third class represents the first reported occurrence of such an extreme number of pseudogenes related to an enzyme-coding gene and suggests that a dramatic amplification event took place between 15 and 25 million years ago.