When a constant number of peripheral human blood mononuclear cells (MC) are seeded in an agar overlayer on top of a cell free underlayer supplemented with 100 microgram phytohemagglutinin (PHA), the number of colonies formed is dependent on cell concentration with respect to both the overlayer and total culture volumes. These results support the view that during PHA-induced colony formation cell cooperation between T-colony forming cells and a population of cooperating cells (CC) is mediated by diffusible soluble mediators. They also indicate that the effect of such mediators is dependent on concentration and not only on total amount in the culture. With respect to contradictory evidence on the nature of CC the possible heterogeneity of the CC population has been explored using the mathematical approach developed by Copplesson and Michie to investigate cell cooperation during an in vitro immune response. As a result, the slopes of the regression lines obtained by plotting the logarithms of the number of colonies obtained against the logarithms of cell inocula suggest the possibility of at least 3 interacting cell populations during colony formation, and thus the heterogeneity of cooperating cells. It is therefore hypothesized that T-cell colony formation may be under the control of a fairly complex system of mediators exchanged by different types of CC rather than by one specific colony stimulating factor as described during granulocyte or macrophage colony growth.