The relationship between the requirement for Ia+ accessory cells (IaAC) and the differentiation of the precursor of CTL (PCTL) in the primary in vitro induction of allogeneic CTL was studied. The following observations were made PCTL can complete the antigen recognition step in the absence of IaAC. This process probably does not involve cell division. The sensitized PCTL require additional signals, which are provided through an IaAC-dependent process to differentiate into the effector CTL. IaAC from any of the responder, the stimulator, or even the third-party strains can fulfill the requirement. However, IaAC of the responder strain failed to generate the helper signal in the absence of allogeneic antigens. The cellular events taking place in the induction of CTL are discussed.