High levels of material with lymphotoxin- (LT) like activity, cytolytic for L-929 cells in vitro, were rapidly released by lymphoid cells obtained from rat, guinea pig, hamster, and rabbit after co-culture for 7 to 10 hr with lectin-coated L-929 cells in vitro. These supernatants were all rapidly fractionated under similar conditions by molecular sieving, ion exchange chromatography, and polyacrylamide gel electrophoresis, and the results were compared to those previously obtained for human and murine LT molecules. The activity in all supernatants was separated by gel filtration into multiple m.w. classes that were strikingly similar to those previously assigned to human and murine LT molecules, i.e. complex (>200,000 d), α (70 to 150,000 d), β (35 to 50,000 d), and γ (12 to 20,000 d). The α m.w. class was resolved on Ultrogel AcA 44 into two distinct m.w. forms, termed α heavy (α(H)) (110 to 150,000 d), and α light (α(L)) (70 to 100,000 d). All supernatants contained LT forms of the Cx, α(H) and α(L) m.w. classes. The smaller β and γ m.w. forms were not as evident in supernatants induced in this fashion. The percentage of activity in each m.w. class varied, but the majority of activity was generally due to α(H) and α(L) forms. However, the relative percentage of activity in any given m.w. class was dependent upon the ionic strength of the separating column buffer. The actual molecular dimensions of materials within the α m.w. class varied somewhat between species, however, they did not vary within the smaller m.w. β and γ forms when detected. The α(H) and α(L) m.w. forms could each be further resolved into multiple charge subclasses. Although the α(H) and α(L) forms in guinea pig and rabbit have been shown by immunologic and/or physical data to be related forms, they can be distinguished by charge from one another. This evidence supports the concept that LT molecules detected in supernatants from experimental animals, although heterogeneous, represent a system of related subunits similar to that defined for the human and mouse. Moreover, this system of cell toxins appears to be conserved in evolution and has common features in each of the animals examined in this study.