The purpose of this investigation was the induction of clonal proliferation of PHA-stimulated normal human lymphocytes using a two-layer soft agar technique. Essential conditions for colony formation include preceding sensitization of lymphocytes with PHA, and continuous presence of PHA in the soft agar culture. Two types of colonies developed: large colonies which appeared 3-4 days after seeding and comprised, after 5-6 days, 200-500 cells, and small colonies which were seen after 6-7 days of culture, resulting in production of 50-150 cells. Morphological study showed that all cells were blast-like and the mitotic index exceeded that in liquid medium by a factor of 50. Comparison between the number of colonies developing from cultured bone marrow and spleen cells with those from peripheral blood showed that, in proportion to the number of lymphocytes seeded, a larger number of colonies developed from bone marrow cells and a lower number of colonies developed from spleen cells. The time required for sensitization of lymphocytes in liquid medium with PHA was found to be no less than 12 hours. The greatest number of colonies appeared when the optimal concentration of PHA was placed in the lower agar layer. A linear relation between the number of cells seeded and the number of resulting colonies was found. One out of 2 X 10(3) or 3 X 10(3) lymphocytes in peripheral blood has the potential to develop as colony. The rosette-forming ability and morphological identification of the cells suggest that the colonies are composed of T lymphocytes.