The restoration of sensitivity to azathioprine of athymic (nu/nu) mouse splenic rosette-forming cells (RFC) has been used by others to assay thymic hormone. These previous studies have led to the conclusion that a major factor in the development of autoimmunity in New Zealand (NZ) mice and patients with systemic lupus erythematosus (SLE) is the premature loss of thymic hormone. We have studied the mouse rosette assay and find that poly A-Poly U and single stranded DNA, in addition to thymosin, restored azathioprine sensitivity to nu/nu RFC. Thus, other substances may mimic thymic hormone, perhaps via the cyclic AMP system. Furthermore, antibodies to T cells were found to interfere with the assay. Sera from old NZ mice which contain naturally occurring thymocytotoxic antibodies (NTA) failed to restore azathioprine sensitivity. Small quantities of NTA prevented normal sera from functioning in the assay. We conclude that the role of thymic hormone(s) in the development of autoimmunity in NZ mice and patients with SLE must be completely reevaluated.