A number of conflicting proposals have been put forward as to the roles and identities of cooperating cells involved with human T-lymphocyte colony formation. To resolve this conflict, the principal cellular interactions of human T-lymphocyte colonies were analyzed using a two-step culture technique. Through mathematical analysis of cell dose-response curves the requirement for at least two populations of cooperating cells in addition to the colony-forming cell was demonstrated. By removing adherent cells prior to the first but not the second step, colony formation was inhibited, which suggests a restricted role for these cells during the early stages of colony formation. The dependence of the second step on interleukin 2 (IL2) availability implied that IL2-producing cells composed the other population of cooperating cells. We therefore propose that colony formation can be described by the two interdependent reactions of activation (T----T') and proliferation (T'----nT'). The first reaction requires an adherent cell (monocyte) population that together with PHA provides the necessary signals for cellular activation. Cell contact is needed to allow the expression of this adherent cell or interleukin 1 ( IL1 ) activity. As a result of these signals at least two subpopulations of lymphocytes are activated, the T-lymphocyte colony-forming cells themselves ( TLCFC ) and a population of cooperating T cells. These activated T cells interact during the second or proliferative reaction. In response to PHA the cooperating T cells release IL2, thereby enabling the activated TLCFs to proliferate into colonies. Thus for PHA-induced T-colony formation, TLCFCs required the cooperation of adherent cells for the activation reaction and IL2-producing T cells for proliferative reaction.