Determination of the immunoreactive fraction (IF) of radiolabeled monoclonal antibodies (MAb) is essential to the understanding of the effects of radiolabeling and subsequent target-specific tumor localization. There has been generally no accepted method of determining the IF of MAbs. The conventional method is based on a radioimmunoassay technique in which the fraction of radiolabeled MAb bound to antigen under conditions of "antigen excess" is determined. Lindmo et al. introduced a modified method in which the IF is determined by extrapolation to conditions representing "infinite antigen excess." Although the Lindmo method, in principle, is insensitive to experimental parameters, it does not always provide a reliable estimate of IF. We, therefore, evaluated an alternate method in which percent cell bound fraction is measured under conditions of fixed antigen concentration and various dilutions of radiolabeled MAb. We developed a mathematical equation to estimate immunoreactivity. J591 MAb specific for prostate-specific membrane antigen was radiolabeled with (111)In, (90)Y and (177)Lu to specific activities of 1-20 mCi/mg. We compared the effect of several experimental conditions on the determination of IF using all three different methods. The Lindmo method requires careful optimization of experimental conditions for each radiolabeled MAb. The alternate method, based on a fixed antigen concentration, appears to be practical and may provide a more reliable measure of immunoreactivity.