ABSTRACIFour aminopeptidases (APs) were separated using native polyacrylamide gel electrophoresis of cell-free extracts and the stromal fractions of isolated chloroplasts prepared from primary barley (Hordeum vulgare L., var Numar) leaves. Activities were identified using a series of aminoacyl-,B-naphthylamide derivatives as substrates. AP1, 2, and 3 were found in the stromal fraction of isolated chloroplasts with respective molecular masses of 66.7, 56.5, and 54.6 kilodaltons. AP4 was found only in the cytoplasmic fraction. No AP activity was found in vacuoles of these leaves. It was found that 50% of the L-Leu-#-naphthylamide and 25% of the L-Arg-ji-naphthylamide activities were localized in the chloroplasts. Several AP activities were associated with the membranes of the thylakoid fraction of isolated chloroplasts. API, 2, and 4 reacted against a broad range of substrates, whereas AP3 hydrolyzed only LArg-B-naphthylamide. Only AP2 hydrolyzed L-Val-a-naphthylamide. Since AP2 and AP3 were the only ones reacting against Val-#-naphthylamide and Arg-8-naphthylamide, respectively, several protease inhibitors were tested against these substrates using a stromal fraction from isolated chloroplasts as the source of the two APs. Both APs were sensitive to both metallo and sulfhydryl type inhibitors. Although AP activity decreased as leaves senesced, no new APs appeared on gels during senescence and none disappeared.Soluble proteins are rapidly degraded or mobilized in the early phases of leaf senescence. Chloroplast proteins are among the first to be degraded, and chloroplast constituents disappear faster than the chloroplast organelles (10,13,28). This suggests that chloroplasts contain proteolytic activities capable of degrading their protein constituents. Such proteolytic activities in chloroplasts have, in fact, been found. Isolated chloroplasts from barley (3) and soybean (19) were capable of degrading RuBPCase4; an ATP-dependent proteolytic activity was located in thylakoids from pea chloroplasts (8, 11), and one endoprotease and three APs were found in the stromal fraction of pea chloroplasts (9); endoproteolytic activity against RuBPCase was found in the stromal fractions of chloroplasts from barley leaves (22,23) Nettleton et al. (17) and Tang and Huffaker (23) found proteolytic activities associated with the thylakoid fractions of chloroplasts from wheat and barley leaves, respectively, capable of degrading RuBPCase. In wheat, 50% of the AP activity was localized in chloroplasts with the remainder in the cytoplasm (30).To better understand the degradation ofchloroplastic proteins, it is necessary to identify and characterize the proteases and their subcellular localization. Recent information shows that the in vivo half-life of some bacterial proteins is a function of their amino terminal amino acid residue (2). If future work shows this applicable to plants, APs may be important in the degradation of chloroplast protein. In this paper, we have identified four APs from barley leaves that are localized in both...