A new one-step ELISA using two monoclonal antibodies specific for distinct epitopes of the free form of protein S (ELISA-m) has been developed for the direct measurement of free protein S in untreated plasma. This assay has been compared with the classic method using polyclonal antibodies to protein S (ELISA-p). The latter method has the drawback of requiring PEG precipitation of plasma which is time-consuming, difficult to perform with accuracy and therefore poorly reproducible in most laboratories. Results of both ELISAs were compared with those of a functional assay. In 30 normal subjects, there was an excellent correlation between ELISA-m and ELISA-p (r = 0.95) as well as between ELISA-m and the functional assay (r = 0.96). In twelve patients with a congenital deficiency, the levels of free protein S antigen were similarly decreased with ELISA-m and ELISA-p and in good agreement with those of protein S activity. In 20 patients with miscellaneous inflammatory diseases, the levels of free proteins S were normal with good correlation between both ELISAs and PS activity, despite high levels of C4bBP-protein S complexes. As expected, in 15 dicoumarol-treated patients, there was a significant and parallel decrease of free protein S antigen with both ELISAs, with even lower levels of protein S activity. In 14 patients with liver cirrhosis, the mean values for free protein S antigen were normal using both assays, but with wide extreme values, whereas protein S activity was significantly lower.(ABSTRACT TRUNCATED AT 250 WORDS)