Three monoclonal antibodies (mAbs), termed SN2, SN2a and SN2b, were used in the present work to study a human T-cell leukemia-associated cell surface glycoprotein, GP37. Strong specificity of mAbs SN2, SN2a and SN2b for T leukemia cells was demonstrated by radioimmunoassay and fluorescence-activated cell sorter (FACS) analysis. GP37 was not detected on normal human peripheral blood lymphocytes, purified normal T-cells, normal thymocytes nor normal bone marrow cells. Furthermore, GP37 was barely detectable on phytohemagglutinin (PHA)- and Concanavalin A (Con A)-activated T-cells. The results indicate clinical utility of these mAbs. Competitive binding experiments show that the epitopes recognized by SN2 and SN2a are sufficiently close to each other to allow complete reciprocal inhibition of binding whereas the epitopes recognized by SN2 and SN2b are less close to allow only partial reciprocal binding inhibition. The biochemical nature of antigenic determinants defined by these mAbs was studied by treating T leukemia cells with trypsin, chymotrypsin, thermolysin, neuraminidase and mixed glycosidases. The results suggest that the antigenic determinants defined by these mAbs all consist of the protein moiety of the glycoprotein GP37. No significant antigenic modulation was observed when T leukemia cells were reacted with SN2. In a sequential immunoprecipitation experiment, a 125I-labeled leukemia antigen preparation was first treated with a rabbit anti-T leukemia antiserum. The latter had been prepared by immunizing a rabbit with a partially purified human T leukemia antigen preparation and showed a good specificity for T leukemia cells. Subsequent treatment of the labeled antigen preparation with SN2 showed that SN2 antigen had been precleared. Thus, both mouse mAb SN2 and the rabbit anti-T leukemia antiserum react with the same GP37 molecule.