The parameters of the secondary in vitro generation of syngeneic CTL by solubilized tumor antigens have been investigated. With PM and SPM, stimulation was achieved over a 10-fold antigen concentration, 5 microgram/ml to 0.5 microgram/ml, and with whole cells the optimum R/S cell ratio was 80:1 to 100:1. Assuming a 50-fold enrichment of plasma membrane antigens over cells by purifying the PM, this observation suggests that cells are at least 200 times more efficient than PM. A specific tumor antigen that induces CTL in vitro was solubilized with sodium deoxycholate and fractionated on Ultrogel AcA34 followed by affinity chromatography on Lens culinaris lentil lectin-Sepharose 4B column. The purification scheme was shown to have enriched for the specific CTL-inducing activity. The specificity of the induction and effector phases of the reaction was assessed and the data clearly indicate that the reactions were specific. Furthermore, we ruled out the possibility that the induction of CTL by soluble tumor antigen in a secondary reaction could be due to the nonspecific induction of helper activity, which then caused a polyclonal expansion of memory cells.