The requirement for cell division and expression of new genes was examined in the primary and secondary mouse mixed leukocyte culture (MLC). Hydroxyurea (HU) was used to block DNA synthesis and cell division, and 5-bromo-2'-deoxyuridine (BUdR) was used to probe for the expression of new cell-specific genes. In the primary MLC, inhibition of DNA synthesis and cell division by HU almost totally suppressed the generation of initial, target-specific cytotoxicity. When HU was washed out of the cultures, cytotoxicity was generated after a lag time approximately equal to the period of treatment with HU. The rate of development and maximal value of cytotoxicity in HU-reversed cultures was identical to untreated controls, suggesting that the inhibition was not due to a nonspecific lethal effect of the drug. Development of initial cytotoxicity in primary MLC was similarly suppressed by levels of BUdR 25 to 75-fold below the levels of this drug having nonspecific mutagenic effects in lymphocytes, indicating that development of cytotoxicity was also dependent on the expression of a new genetic program. In the secondary MLC, regeneration of both DNA synthesis and cytotoxicity was apparent 12-15 h after re-exposure to initial stimulating antigen. In this reaction, however, generation of cytotoxicity was insensitive to both HU and BUdR. Thus, the cytotoxic program developed in the primary MLC appears to be genetically stable through the production of effector memory cells, and into regeneration of fully cytotoxic memory cells in secondary MLC.