Protein S (PS) possesses a sex-hormone-binding globulin (SHBG)-like domain in place of the serine-protease domain found in other vitamin K-dependent plasma proteins. This SHBG-like domain is able to bind a complement fraction, C4b-binding protein (C4b-BP). To establish whether the PS SHBG-like domain can fold normally in the absence of other domains, and to obtain information on the specific functions of this region, we expressed the PS SHBG-like domain alone or together with its adjacent domain EGF4. The folding of the two recombinant modules was studied by analyzing their binding to C4b-BP. The apparent dissociation constants of this interaction indicated that both recombinant modules adopted the conformation of native PS, indicating that the PS SHBG-like region is an independent folding unit. We also obtained the first direct evidence that the SHBG-like domain alone is sufficient to support the interaction with C4b-BP. In addition, both recombinant modules were able to bind Ca2+ directly, as shown by the migration shift in agarose gel electrophoresis in the presence of Ca2+, together with the results of equilibrium dialysis and the functional effect of Ca2+ on the C4b-BP/PS interaction, confirming the presence of one Ca2+ binding site within the SHBG-like domain. Neither recombinant module exhibited activated protein C (aPC) cofactor activity in a clotting assay, suggesting that the PS SHBG-like region must be part of the intact molecule for it to contribute to aPC cofactor activity, possibly by constraining the different domains in a conformation that permits optimal interaction with aPC.