An improved purification procedure of human aldehyde dehydrogenase (EC 1.2.1.3) isoenzymes E1 and E2 is presented. This procedure employs only three chromatographic steps to produce homogeneous E1 and E2 isoenzymes at 60% overall yield. The isoenzymes have been tested for homogeneity by electrophoresis of native and denatured species, specific activity determinations following rechromatography, as well as mapping of tryptic and CNBr fragments. Total SH group analysis has also been done on each isoenzyme. The results show that both isoenzymes are homogeneous. Similarities between E1 and E2 isoenzymes are noted in the mobility of about 40% of tryptic fragments, total SH content, and the mobility of two CNBr fragments. The results also show considerable structural differences between the isoenzymes in that CNBr maps show fragments from E1 and E2 of different molecular weight and about 60% of tryptic fragments migrate to distinct locations. Only one of SH-containing tryptic fragments migrates to the same location in both isoenzymes. E1 and E2 each consist of subunits which migrate as single bands in both sodium dodecyl sulfate (SDS) and urea electrophoresis. While the mobility of E1 and E2 subunits in SDS gels is similar, it is different in urea, showing that subunits of E1 are distinct from those of E2 and that the isoenzymes do not share subunits. Structural similarity between isoenzymes must, therefore, result from sequence similarity within regions of distinct polypeptide chains composing E1 and E2 molecules. The results presented offer a simplified procedure for preparation of the homogeneous isoenzymes; they also suggest that E1 and E2 are products of distinct genes which probably diverged from a common genetic ancestor through gene duplication and compartmentation of the cell.