In principle, equilibrium analytical ultracentrifugation (AU) can be used to quantify the binding stoichiometry and affinity between small-molecule ligands and proteins in aqueous solution. We show here that heteromeric binding constants can be determined using a data-fitting procedure which utilizes a postfitting computation of the total amount of each component in the centrifuge cell. The method avoids overconstraining the fitting of the radial concentration profiles, but still permits unique binding constants to be determined using measurements at a single wavelength. The computational program is demonstrated by applying it to data obtained with mixtures of a 500-Da molecule and interleukin-2, a 16-kDa protein. The 1:1 binding stoichiometry and heteromeric dissociation constants (K(ab)) determined from centrifuge data at two different wavelengths are within the 4-9 microM range independently determined from a functional assay. Values for K(ab) have been obtained for ligands with affinities as weak as 500 microM. This AU method is applicable to compounds with significant UV absorbance (approximately 0.2) at concentrations within approximately 5- to 10-fold of their K(ab). The method, which has been incorporated into a user procedure for IgorPro (Wavemetrics, Oswego, OR), is included as supplementary material.