Molecules with LT activity which can be identified in supernatants from PHA stimulated human lymphocytes by lysis of murine L-929 cell in vitro are heterogeneous. They can be separated by MW on sephadex or ultrogel columns into four separate classes: (a) complex (>200,000 daltons); (b) α (70-90,000 d); (c) β (25-50,000 d); and (d) γ (10-20,000 d). The amount of activity in a supernatant due to each class varies but is approximately: Cx-5 to 20%, α-40 to 60%, β-20 to 40%, and γ-0 to 10%. These classes differ one from another in their stability and kinetics of appearance in culture. Furthermore, they may aggregate together with the complex class under conditions of low ionic strength. Each class, except γ, can be further separated into subclasses by ion exchange chromatography and polyacrylamide gel electrophoresis. Alpha class can be separated on DEAE into the three subclasses, termed, α1 (rf 0.25), α2 (rf 0.37), and α3 (rf 0.50). Alpha2 subclasses can be further separated on phosphocellulose at pH 6.6 into α2a (rf 0.37) and α2b (rf 0.30). However, α2 contains additional subclasses, which were resolved on PC columns at pH 5.5. Beta class activity can be resolved by DEAE and PAGE into two subclasses, termed β1 (rf 0.28) and β2 (rf 0.49). Gamma class activity was not studied, because of its instability. The complex class of LT activity is a macromolecular aggregate greater than 200,000 daltons which appears to contain smaller MW LT class(es). This study demonstrates that materials with LT activity in supernatants from PHA activated human lymphocytes in vitro are very heterogeneous. © 1978.