Hybridoma clones producing monoclonal antibodies specific for staphylokinase were isolated. A competitive assay revealed that the monoclonal antibodies studied could be divided into at least two groups. Representatives of these groups, AS22 and B3E6, recognized quite different epitopes on staphylokinase. This finding led us to develop an assay system for the quantitative analysis of staphylokinase by enzyme-linked immunosorbent assay using AS22 as the capturing antibody and biotinylated B3E6 as the "detector". The lower limit of sensitivity of the assay was 20 pg of staphylokinase per ml. The assay exhibited good reproducibility, with values of 5.8 and 3.8% for the intra- and inter-assay coefficients of variation, respectively. Staphylokinase could be assayed in the presence of human plasma when the plasma was diluted more than 320-fold, and the measurement was unaffected by the presence of physiological concentrations of human plasminogen. Hence, this assay was considered useful for the detection and quantification of staphylokinase in clinical samples.