Originally developed as a method for estimating the solid-phase concentration of an adsorbed protein, sequential enzyme-linked immunosorbent assay (ELISA) analysis (P. A. Underwood and J. G. Steele (1991) J. Immunol. Methods 142, 83-94) is also useful for assessing other aspects of protein-surface interactions without radioactively labeled reagents. The method provides a way to compare protein adsorption on various microwell surfaces and to track adhesion of the protein throughout an assay's wash and incubation steps. In cases where adsorption complies with Langmuir-type binding and protein desorption is minimal, sequential ELISA data can be analyzed with a mathematical model to quantitatively estimate the microwell's protein-binding capacity. Even for protein-surface interactions where the mathematical model is not appropriate, characteristics of sequential ELISA binding data qualitatively differentiate between initial adsorption with subsequent desorption and poor initial adsorption of the protein. Using this method, we analyzed the binding of three proteins (rabbit immunoglobulin G, bovine serum albumin, and horse spleen ferritin) to four types of polystyrene microwell surfaces (Immulons 1, 2, 3, and 4). In addition, we examined differences in protein adhesion to the four surfaces when Tween 20 was omitted from the assay's wash buffer or when fewer washes were used.