SUMMARYRats received a single high dose of cyclophosphamide (Cy) (150 mg/kg). followed 48 h later (on day 0) by immunization with a T cell-dependent soluble antigen, ovalbumin in Freund's complete adjuvant (FCA). The effect of this treatment on lymphoid cell subpopulalions in the spleen, natural killer (NK) cell and interieukin-2 (IL-2) induced lymphokine-activated killer (LAK) cell activity was examined. Cy (with and without ovalbumin) caused a large relative increase (by day 14) in splenic OX 8+, OX 19-cells with NK morphology. A marked relative increase in fresh NK cell activity was noted after Cy+ovalbumin, but not consistently after Cy alone. Elevated NK activity was Cy dose- and time-dependent, was evident within 7 days post Cy/ovalbumin and persisted for at least 28 days. Pooled splente mononuclear cells (MNC), obtained 14 days after Cy/ovulbumin, lost all cytolytic activity against YAC-I cells when cultured in the absence of human recombinant IL-2 (rIL-2). In contrast, similarly maintained cells from normal rats displayed NK activity higher than normal‘fresh’levels. Upon culture in medium containing 500 U/ml rIL-2, however,‘augmented’NK. activity was equivalent, on a per-cell basis, in both normal and Cy/ovaibumin-pretreuted groups. LAK activity generated in vitro (i.e. against NK.-resistant target cells) was significantly lower in the latter group, and the overall yield of cells was reduced. By day 21 after Cy/ovaibumin, augmented NK activity was significantly greater lhan controls, on a per-cell and total culture yield basis. Moreover, LAK activity was now similar between groups. It is concluded that the chemotherapy/immunization protocol which we have used can greatly enhance NK activity in vitro and that these cells are responsive to induction of LAK activity by IL-2 in ritro.