Light modulation of chloroplast glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) has been investigated. Complete activation of NADPH-dependent activity is achieved at 25 W-m -2 photosynthetically active radiation in spinach (Spinacia oleracea L.) and 100W'm -2 in maize (Zea mays L.) leaves. Light activation is stronger in spinach (fivefotd on average) than in maize (twofold), which shows higher "dark" activity. The NADH dependent activity does not change appreciably. Several substrate activators can simulate in vitro the light effect with recovery of latent NADPH-dependent activity of spinach enzyme, but they are almost inactive with maize enzyme. A mixture of activators has been devised to fully activate the spinach enzyme under most conditions. The NAD(P)-GAPDH protein can be resolved by rapid gel filtration (fast protein liquid chromatography) into three conformers which have different molecular masses according to the light conditions. Enzyme from darkened leaves or chloroplasts, or dichlorophenyl-l,l-dimethylurea-treated chloroplasts is mainly a 600-kDa regulatory form with low NADPH-dependent activity relative to NADH-activity. Enzyme from spinach leaves or chloroplasts during photosynthesis is mainly a 300-kDa oligomer, which along with the 600-kDa form also occurs in leaves of darkened maize. The conformer of illuminated maize leaves is mainly a 160-kDa species. Results are consistent with a model of NAD(P)-GAPDH freely interconvertible between protomers of the 160-kDa (or 300-kDa intermediate) form with high NADPH-activity, produced in the light by the action of thioredoxin and activating metabolites (spinach only), and a regulatory 600-kDa conformer Abbreviations: AEM=activator equilibrium mixture; Chl= chlorophyll; DCMU=dichlorophenyl 1,1-dimethylurea; DTT= dithiothreitol; FPLC=fast protein liquid chromatography; NAD(P)-GAPDH=glyceraldehyde 3-phosphate dehydrogenase, NAD(P)-dependent; PAR = photosynthetic active radiation; PGK=phosphoglycerate kinase; Tricine=N-tris(hydroxy-methyl) methyl-glycine Correspondence to: V. Valenti; FAX: 39(75)5856425 with lower NADPH-activity produced in darkness or when photosynthesis is inhibited. This behavior is reminiscent of the in-vitro properties of purified enzyme; therefore, it seems unlikely that NAD(P)-GAPDH in the chloroplast is part of a stable multienzyme complex or is bound to membranes.