Specific helper and suppressor cells were induced in vitro with low and high doses, respectively, of protein antigen in Marbrook cultures. On different days of culture the cells from helper and suppressor cultures were size-fractionated by 1-g velocity sedimentation and the different fractions tested for specific activity in secondary co-operative cultures after challenge with protein-hapten conjugate. We observed a change in the size distribution of both helper and suppressor cells during their in vitro differentiation; the first helper and suppressor cell to be detected is a large (blast) cell, whereas later both help and suppression is mediated by medium- and small-sized lymphocytes. A difference between helper and suppressor cells with regard to their relative dependence on DNA synthesis and proliferation during their induction was also seen. Although both DNA synthesis and divisions are needed for the optimal generation of both types of cells, some suppressor activity was still induced in cultures treated with mitosis-arresting concentrations of demecolcine (Colcemid). Treatment of helper cell cultures with Colcemid or with 5-bromodeoxyuridine plus light completely abolished the help.