To study the mechanisms that regulate the activity of interleukin 2 (IL 2) and possibly limit its activity, we have examined normal mouse serum for their ability to inhibit IL 2-mediated proliferation of a cloned IL 2-dependent cytotoxic T lymphocyte line (CTLL). Normal mouse serum contains a factor capable of inhibiting IL 2 dependent proliferation of CTLL cells. This factor is absorbed with IL 2-dependent cells, but not with IL 2 molecules. Decreased activity of the inhibitor is observed in serum from autoimmune-prone mice such as NZB and NZBWF 1 hybrid mice. The results suggest that the serum IL 2 inhibitor may play an important role in the in vivo regulatory mechanism of IL 2 activity and that lack of the inhibitor may be associated with aberrant immune functions.