Southern blot analysis of human genomic DNA hybridized with a coding region aldolase A cDNA probe (600 bases) revealed four restriction fragments with EcoRI restriction enzyme: 7.8 kb, 13 kb, 17 kb and greater than 30 kb. By human-hamster hybrid analysis (Southern technique) the principal fragments, 7.8 kb, 13 kb, greater than 30 kb, were localized to chromosomes 10, 16 and 3 respectively. The 17-kb fragment was very weak in intensity; it co-segregated with the greater than 30-kb fragment and is probably localized on chromosome 3 with the greater than 30-kb fragment. Analysis of a second aldolase A labelled probe protected against S1 nuclease digestion by RNAs from different hybrid cells, indicated the presence of aldolase A mRNAs in hybrid cells containing only chromosome 16. Under the stringency conditions used, the EcoRI sequences detected by the coding region aldolase A cDNA probe did not correspond to aldolase B or C. The 7.8-kb and greater than 30-kb EcoRI sequences, localized respectively on chromosomes 10 and 3, correspond to aldolase A pseudogenes; the 13-kb EcoRI sequence localized on chromosome 16 corresponds to the aldolase active gene. The fact that the aldolase A gene and pseudogenes are located on three different chromosomes supports the hypothesis that the pseudogenes originated from aldolase A mRNAs, copied into DNA and integrated in unrelated chromosomal loci.