Three mouse monoclonal antibodies (RFF-VII/1, RFF-VII/2, and RFF-VII/3) which bind specifically to different epitopes on human factor VII antigen were raised. Two of the antibodies, RFF-VII/1 and RFF-VII/2, bound strongly to factor VII antigen (VII:Ag), but only RFF-VII/1 and RFF-VII/3 were potent inhibitors of factor VII coagulation activity (VII:C). RFF-VII/1 and RFF-VII/2 were used in a one step, double monoclonal immunoradiometric assay for VII:Ag. This was highly reproducible and detected as little as 0.05U/dl VII:Ag. Values for VII:Ag obtained for plasma samples from normal subjects (n = 20), patients with liver disease (n = 20), patients treated with warfarin (n = 20), and those congenitally deficient in factor VII (n = 7) correlated very well (r = 0.96) with data obtained in a radioimmunoassay using polyclonal rabbit antiserum to factor VII. This simple and sensitive monoclonal antibody based assay offers a convenient method for the detection of VII:Ag in various disease states.