Monoclonal antibodies to human protein S have been prepared using established hybridoma technology. One antibody was isolated that binds protein S only when Ca2+ is present; others bind antigen equally well in the presence or absence of EDTA. Other antibodies display a diminished affinity for protein S in the presence of EDTA. Purified immunoglobulins from cell lines displaying Ca2+ dependence were immobilized and used to purify protein S from fractions obtained by barium precipitation of citrated plasma, ammonium sulfate fractionation, and chromatography on diethylaminoethanol (DEAE)-Sephadex and dextran sulfate agarose. Essentially homogeneous protein S was isolated from the barium-citrate-adsorbed, 35% ammonium-sulfate-soluble proteins using a totally Ca2+-dependent antibody and EDTA elution. Protein S and several substances of higher mol wt were bound directly from plasma by a partially Ca2+-dependent antibody and were eluted partially with EDTA and NaCl and finally with NaSCN. The largest and most abundant of the high mol wt materials is likely protein S-complement C4b-binding protein complex. The immunoaffinity-isolated protein S was found to be indistinguishable from conventionally isolated protein S in terms of activity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) mobility, and by high-performance liquid chromatography (HPLC). These studies establish reagents that can probe the structure of protein S and isolate protein S in its free and complexed forms.