To determine the role of the thiol ester in the folding of human alpha 2-macroglobulin (alpha 2M) in the active conformation, we have characterized a recombinant variant of alpha 2M, C949S, expressed in baby hamster kidney cells, that lacks the thiol ester-forming cysteine. C949S alpha 2M behaves like methylamine-treated plasma alpha 2M, with correctly formed inter-subunit disulfide bridges, non-covalent association of covalent dimers to form tetramers, and exposure of the receptor binding domain, but an inability to inhibit proteinases, and inaccessibility of the bait regions to proteolysis. We concluded that correct folding of monomers or their association to give tetrameric alpha 2M does not require a pre-formed thiol ester. Active alpha 2M may form in vivo by a two-step process involving initial folding to give a structure resembling that of C949S alpha 2M followed by thiol ester formation and a conformational change that gives the native active state.