A luminol-enhanced chemiluminescence (CL) assay was developed to evaluate type III group B streptococcal opsonins. Sera from 11 adults that contained high (greater than 25 microgram/ml), intermediate (3-24 microgram/ml), or low (less than 2 microgram/ml) antibody concentrations were assessed by CL, indirect immunofluorescence (IF), and bactericidal assays. Total CL activity correlated with antibody concentration (P less than 0.005), IgG- and C3-IF values, and phagocytic and bactericidal indices. Heat-stable CL activity correlated with antibody concentration (P less than 0.0001) and was related to IgG-IF values and phagocytic indices. Significant bactericidal activity was demonstrated in all sera containing greater than 2 microgram of antibody/ml, but no bactericidal activity was observed in heat-inactivated sera. Adsorption of heated sera with type III group B Streptococcus abolished CL activity, whereas CL values of MgEGTA-chelated sera were approximately 80% of untreated sera. Thus, type-specific antibody in addition to heat-labile serum factors, including those of the alternative complement pathway, contribute to opsonization of type III group B Streptococcus.