Acyl carrier protein (ACP) contains an essential sulfhydryl group in its phosphopantetheine prosthetic group. We have investigated the state of this sulfhydryl in developing and mature spinach seed (Spinacia oleracea). Seed extracts were separated on sodium dodecyl sulfate or native polyacrylamide gels, blotted to nitrocellulose, and probed with antibodies raised against spinach ACP-I. In extracts of mature seeds prepared with reducing agents, ACP-11 migrated as a single major band, whereas extracts prepared without reducing agents gave two major bands. The additional band was identified as a conjugate of ACP-11 to glutathione (ACP-S-S-G) on the basis of its sensitivity to reducing agents and its comigration with standards in both native and sodium dodecyl sulfate gel electrophoresis. In developing spinach seeds ACP-1l exists primarily in its free sulfhydryl form or as acyl derivatives, with essentially no ACP-S-S-G present. During later stages of seed development, as seed water content declines, ACP-S-S-G accumulates to approximately 50% of the total ACP. Seed imbibition results in a rapid decline in ACP-S-S-G levels. The ACP-S-S-G:ACP-SH ratio of seeds during storage was found to be a function of seed water content and this could be manipulated by controlling the relative humidity under which the seeds were stored. We speculate that conjugation of ACP to glutathione protects the ACP from sulfhydryl oxidative damage in dry seeds.The synthesis of fatty acids in plants occurs mainly in plastids and is catalyzed by a series of enzymes together with the protein cofactor, ACP3 (6). In plants, ACP is a small (ca. 9000 D), acidic cofactor which plays a central role in plant lipid metabolism (13). A phosphopantetheine prosthetic group is attached to a seine residue near the middle of the polypeptide and is the site at which acyl chains are esterified during the reactions of fatty acid assembly. In addition to the six enzymatic steps of fatty acid synthesis, ACP also participates in reactions catalyzed by stearoyl-ACP desaturase, two acyl-ACP dependent chloroplast acyltransferases which acylate glycerol-3-phosphate and oleoyl-ACP thioesterase, which