Thioguanine derivatives with reactive ester groups at positions 6, 7, or 9 of the purine ring were synthesized and coupled to a protein carrier. The purified protein derivative of tuberculin was used as the carrier for immunizing bacillus Calmette-Guerin primed mice. This led to high antibody titers against the homologously coupled hapten, and spleen cells from the immunized mice were used to produce monoclonal antibodies against thioguanine. All monoclonal antibodies were selected for their ability to recognize free thioguanine and were analyzed for their fine specificity by inhibition experiments with a panel of thiopurine derivates. The specificity of the monoclonal antibodies showed a strong dependence on the coupling position of the thioguanine. Within each group of monoclonal antibodies, raised against one of the three different conjugates, there was a high degree of heterogeneity, with antibodies differing in their binding according to the substitution on the thioguanine analogues used in the inhibition experiments. This panel of antibodies may be used for quantitative assays of thiopurines and their metabolites in patients undergoing treatment with thioguanine, 6-mercaptopurine, and azathioprine.