Acyl carrier protein (ACPSH) functions as the acyl carrier in fatty acid biosynthesis. The acyl moieties are bound to the sole sulfhydryl of the protein located on the 4'-phosphopantetheine prosthetic group. Disulfide-linked dimers of ACPSH were formed by the reaction of ACPSH with acyl-ACP or the mixed disulfide of ACPSH and thionitrobenzoate. The formation of ACP dimers was established by electrophoresis, gel filtration, and sedimentation equilibrium. ACP purified from stationary phase Escherichia coli B cells was found to exist primarily as a mixed disulfide with glutathione. This species was identified by gel electrophoresis amino acid analysis and 31P NMR spectroscopy. A non-denaturing gel electrophoresis system was developed that allows the comparison of the effects of various protein and sulfhydryl modifications on the stability of the ACP protein moiety to pH-induced denaturation. In general, attachment of hydrophilic ligands to the sulfhydryl of ACPSH resulted in less stable protein structures whereas the presence of a hydrophobic thioester resulted in stabilization of the protein conformation. The less stable ACP structures were found to have 31P NMR chemical shifts displaced downfield from ACPSH and the more stable acyl-ACP derivatives were found to have chemical shifts displaced upfield from ACPSH.