Using conditioned media (CM) from phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBL) we observed long-term selective growth of T-cells from normal human donors. This T-cell growth was continuously dependent on addition of a factor called T-cell growth factor (TCGF). The optimal method for preparing highly active CM from single donor PBL involves the addition of mitomycin C-treated B-lymphoblastoid cell lines to the mixture of PBL and PHA. A number of different cell lines greatly augmented the production of TCGF in 18/18 cases. Preparation of plasma membranes from the Daudi cell line could replace the intact cells in the production of TCGF but those from the cell line, Molt-4, could not. Since the cell surface of Daudi possesses HLA-D antigens but not HLA-A, B, and C, and Molt-4 has HLA-A and B and not HLA-D, it is possible that the Ia antigens (HLA-DRw in man) are important in the release of TCGF. Using this method for growth factor production, an analysis was made concerning the events necessary for lymphocyte activation and the requirements for production and release of TCGF. Removal of PHA 12 h after incubation had no effect on lymphocyte transformation but decreased TCGF release by 90%. In addition, colchicine and cytosine arabinoside inhibited DNA synthesis but had no effect on TCGF release. Little or no TCGF activity was present after cellular protein synthesis was inhibited by puromycin and cycloheximide. These results suggest that TCGF production: a) requires protein synthesis; b) requires binding of the stimulating agent; c) can occur in a non-dividing cell, probably a terminally differentiated T-cell, without the need for cellular proliferation; and d) needs the assistance of an adherent cell which probably is a monocyte-macrophage. The ability to produce TCGF from single human donors will allow better understanding of the nature and action of TCGF.