The thermonuclease of Staphylococcus aureus was inactivated during incubation with filtrates from cultures of Streptococcus faecalis subsp. liquefaciens . Of 53 isolates, 40 produced a thermonuclease-inactivating factor (TIF). The TIF was heat-labile, non-dialyzable and optimally active at 55 C and pH 7.0. These characteristics suggest TIF is a proteolytic enzyme. The common occurrence of TIF in cultures of S. faecalis subsp. liquefaciens may limit the value of the thermonuclease test for foods containing large populations of this organism.