To assess the heterogeneity of T cells activated during the autologous mixed-lymphocyte reaction (AMLR), a cloning procedure based on the soft agar colony assay was developed. Supernatants of allogeneic MLR cultures were used as a source of interleukin 2 (IL-2) to generate two types of colonies: upper and lower colonies. Both types of colonies were expanded in long-term cultures using supernatants of phytohemagglutinin (PHA)-activated lymphocyte cultures. Cloned cells underwent secondary proliferation when stimulated by autologous monocytes, although certain clones also responded to autologous B cells. Most autoactivated clones expressed the serological determinants of HLA-DR, MB, and MT and were OKT3+, OKT4+, OKT8-. They did not induce cytolysis of autologous monocytes, B lymphoblasts, or PHA blasts nor did they express natural killer-like activity toward K562 cells. Several autoactivated clones (irradiated with 500 R) expressed helper activity, shown by an enhancing effect on AMLR proliferation. Furthermore, many irradiated clones were capable of inducing proliferation of autologous T cells in the absence of accessory cells. These observations suggest that autoactivated clones generated from soft agar colonies may interact with autologous T lymphocytes.