Many radioligands used to study neurotransmitter receptors label multiple subtypes or multiple classes of receptors. Frequently, two subtypes or classes of receptors coexist in the same tissue and exhibit only slightly different affinities for a radioligand. A new analytical method has been developed to quantitate the interaction of a slightly selective radioligand with two distinct classes of binding sites. This new approach requires that a series of inhibition experiments with a selective unlabeled ligand be performed in the presence of increasing concentrations of the radioligand. Analysis of the resulting inhibition curves, utilizing simultaneous nonlinear regression analysis techniques, yields estimates of the density of each class of receptor as well as the affinity of each receptor for the labeled and unlabeled ligands. This approach was used to characterize the interaction of [3H]spiroperidol with serotonin 5-HT2 and dopamine D2 receptors. A series of inhibition curves were generated with the 5-HT2 selective antagonist ketanserin using membranes prepared from rat striatum. [3H]Spiroperidol was found to be 23-fold more selective for D2 receptors and 27% of the sites labeled by [3H]spiroperidol exhibited a high affinity for ketanserin and were presumed to be 5-HT2 receptors. Since all of the radioligands commonly used to label 5-HT2 receptors also label other receptors, this method of analysis should be particularly useful in the study of these receptors.