Using fluorescence spectroscopy, we have demonstrated that isolated envelope membranes from mature spinach chloroplasts catalyze the phototransformation of endogenous protochlorophyllide into chlorophyllide in presence of NADPH, but not in presence of NADH. Protochlorophyllide reductase was characterized further using monospecific antibodies (anti-protochlorophyllide reductase) raised against the purified enzyme from oat. In mature spinach chloroplasts, protochlorophyllide reductase is present only in envelope membranes. We have demonstrated that the envelope protochlorophyllide reductase, a 37,000-dalton polypeptide, is only a minor envelope component and is present on the outer surface of the outer envelope membrane. This conclusion is supported by several lines of evidence: (a) the envelope polypeptide that was immunodecorated with anti-protochlorophyllide reductase can be distinguished from the major 37,000-dalton envelope polypeptide E37 (which was identified by monospecific antibodies) only after two-dimensional polyacrylamide gel electrophoresis; (b) the envelope protochlorophyllide reductase was hydrolyzed when isolated intact chloroplasts were incubated in presence of thermolysin; and (c) isolated intact chloroplasts strongly agglutinate when incubated in presence of antibodies raised against protochlorophyllide reductase. These results demonstrate that major differences exist between chloroplasts and etioplasts with respect to protochlorophyllide reductase levels and localization. The presence on the chloroplast envelope membrane of both the substrate (protochlorophyllide) and the enzyme (protochlorophyllide reductase) necessary for chlorophyllide synthesis could have major implications for the understanding of chlorophyll biosynthesis in mature chloroplasts.