The adsorptive characteristics of polystyrene tubes for seven proteins of different molecular weight and ionic charge have been studied. The stability of these adsorbed proteins was monitored throughout the various steps of the amplified-enzyme-linked immunosorbent assay (a-ELISA). Using 1251-1abe1ed proteins, it was observed that each had a characteristic adsorptive behavior not explainable by simple charge differences. The proportion of protein bound to a 65cm* tube surface was independent of the amount added up to an input of about 1000 ng. Binding increased proportionately with temperature and incubation time. Beyond the range of concentrations where the proportion bound was independent of input, i.e., the region of independence, the kinetics of adsorption changed. The amount of protein adsorbed at the upper limit of the region of independence was inversely proportional to molecular weight for all except a-lactalbumin. Quantitative measurement of bound protein using the a-ELISA suggests that beyond the region of independence, protein-to-protein rather than protein-to-polystyrene adsorption occurred. This was suggested by computation using the Stokes radii of the proteins which implied that the amount of protein bound at the upper limit of the region of independence was a layer of protein one molecule thick. The upper limit of the region of independence determines, therefore, the maximum amount of functional antigen for such assays. Once adsorbed, proteins remained stably bound throughout the assay steps. The presence of competing proteins had no effect on the adsorptive characteristics of a given protein provided that the total protein concentration of the mixture was within the range of independence. The results indicate that the adsorptive characteristics of proteins for polystyrene tubes routinely used in solid-phase immunoassays should be taken into consideration when designing and interpreting data obtained with such assays.