An enzyme‐linked immunosorbent assay (ELISA) for rabbit immunoglobulin (Ig) was developed in order to investigate serum levels achieved by therapeutic doses of rabbit antithymocyte globulin (ATG) and their relationship toin vitroserum lymphocytotoxic activity. Twenty renal allograft recipients treated for acute steroid resistant rejection with ATG were studied, where possible, before, during and following their treatment with ATG. During therapy, peak serum levels of rabbit Ig were in the range 20–101μg/ml with one exception of 404μg/ml. After treatment, levels gradually declined to zero within 12 weeks. AH sera with lymphocytotoxic activity were absorbed with platelets and treated with dithiothreitol so that reactivity due to anti‐HLA antibodies and autoantibodies produced by the patient could be differentiated from each other and from serum ATG. Nine patients had detectable serum ATG associated within vitrolymphocytotoxic activity. In two cases the lymphocytotoxicity was attributed solely to ATG; two had only anti‐HLA antibodies; in one patient the lymphocytotoxicity was due to a combination of ATG and anti‐HLA antibodies; two had autoantibodies; and for two, cytotoxicity was initially due to ATG and subsequently to the development of autoantibodies. The serum levels of ATG achieved during treatment could thus be quantified, and the important distinction between anti‐HLA antibodies, autoantibodies and ATG