One of the problems that has plagued the investigation of soluble suppressor T cell factors (TsF) has been the lack of sufficient material for direct assessment of their serological reactivities and biochemical characteristics. In the studies described in this communication, biosynthetically radiolabelled TsF and purified, radio-iodinated TsF were used to address these issues. We found that the cell-associated form of a factor specific for L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT-TsF1) is a 66 kDa protein which exhibits a slightly acidic isoelectric point, but no carbohydrate as determined by enzymatic analysis and lectin-affinity chromatography. The biological analysis of suppression is specific for GAT and the specific activity of GAT-TsF1 is 2 x 109 suppressive units/μg. Direct measurement of the antigen binding capacity of GAT-TsF1 by affinity chromatography demonstrated that it binds to GAT but not an irrelevant antigen, and that only GAT-binding TsF1 is biologically active. Furthermore, GAT-TsF1 can be specifically eluted from GAT-Sepharose with soluble GAT. Lastly, purified GAT-TsF1 binds to polyvalent anti-TsF1 and alloantisera produced against the I region histocompatible cells of the H-2(q) haplotype, the so-called anti-I-J antibodies, but does not bind to immunosorbents containing antisera to irrelevant I-region haplotypes. Thus, these data physically verify the serological characteristics of GAT-TsF1 that were originally defined solely on the basis of biological activity.