Extracts from 13 different angiosperm species (spinach, mustard, pea, bean, tomato, cucumber, pumpkin, maize, sorghum, rye, wheat, oats, barley) were submitted to electrophoresis under nondenaturing conditions and stained for enzyme activities of cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases by a modified tetrazolium test of high sensitivity. Zymograms of the cytosolic enzyme revealed a single band of similar electrophoretic mobility for all but one species, the tomato, which displayed an ordered set of five different bands. In contrast, zymograms of the chloroplast dehydrogenase are highly different, containing between two and five distinct bands of variable electrophoretic mobilities according to the plant species examined. This variability of the native chloroplast enzyme is paralleled by a remarkable interspecific heterogeneity of the enzyme with respect to subunit size and number, as shown by dodecylsulfate electrophoresis of the purified chloroplast enzyme from 11 different angiosperm species. The present data suggest that cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases belong to two separate protein families of different evolutionary rate. While the cytosolic enzyme is probably an extremely conservative protein like the corresponding enzymes from animals, yeast and bacteria, the chloroplast enzyme seems to change rather rapidly during evolution.