The present studies investigate the physicochemical and immunologic properties of cell-lytic molecules released in vitro by nonadherent C57/BL/6 splenocytes or nylon wool-enriched T cell populations activated on monolayers of PHA coated L-929 cells. The findings reveal that cell-lytic molecules released by these lymphoid cells are physically heterogeneous. These molecules can be separated by gel filtration into similar m.w. classes previously observed for human lymphotoxin (LT) molecules. Three major classes, termed complex (Cx) (>200,000 d), α heavy [α(H)] (110 to 140,000 d), α light [α(L)] (60 to 90,000 d), and two minor classes, β at 40 to 50,000 d and γ at 10 to 20,000 d, were observed. Chromatography of supernatants in high ionic strength buffers dissociated Cx and α(H) to the smaller m.w. α(L) form. This evidence suggests that Cx and α(H) MW classes are physically related to the smaller m.w. α(L) class. Fractionation of the α(H) m.w. LT class by DEAE or PAGE resolved these molecules into additional distinct subclasses. Antisera were made against fresh serum-free whole supernatants (anti-WS) or rechromatographed Ultrogel fractions containing α(H) molecules [anti-α(H)]. Anti-α(H) and anti-WS react with all m.w. classes of murine LT molecules, indicating these various forms are immunologically related. These antisera do not react with LT molecules obtained from several other animal species or with 'nonspecific' intracellular toxins, e.g., lysosomal enzymes, present in normal PMN or phagocytic cells. These data indicate that materials with cell-lytic activity present in these culture supernatants are LT molecules, because: a) certain m.w. forms observed are similar to those reported previously, and b) these various m.w. forms are all physically and immunologically interrelated. These studies also indicate that murine LT molecules like human LT molecules are heterogeneous, but appear to comprise a system of subunits, in which the large m.w. form may dissociate into the smaller m.w. forms.