B6AF1 anti-B10.D2 ascites fluid was fractionated by molecular sieving and cation exchange chromatography in an attempt to separate cytoxic and enhancing antibodies. Five fractions were obtained which showed no overlap on analysis with isoelectric focusing. Despite this complete physicochemial separation all fractions contained detectable amounts of 7S IgG1 and 7S IgG2. Furthermore, exhancement of B10.D2 skin grafts in B6AF1 recipients could be induced with all the fractions. Cytotoxic activity was also present in the fractions. This was not only shown in vitro by the cytolysis of B10.02 lymphoid cells in the presence of rabbit complement, but also by the ability of the different fractions to induce hyperacute destruction of B10.02 skin grafted onto B6AF1 mice after i.v. injection together with rabbit complement. Thus, we were unable to separate the alloantiserum in cytotoxic and enhancing fractions by physicochemical means.