In this report, the ontogeny of precursors of T cell growth factor (TCGF)-producing cells in the mouse thymus was investigated using a recently described limiting dilution microculture system. In agreement with previous studies, in the adult thymus TCGF production by cells stimulated by alloantigens was largely the property of the Lyt-2-negative subpopulation. Furthermore, when Lyt-2-negative cells were stained with monoclonal antibody GK-1.5 and sorted according to fluorescence intensity, all precursors of TCGF-producing cells were quantitatively recovered in the GK-1.5-positive subpopulation. During ontogeny, TCGF production by Lyt-2-negative thymocytes was first detectable on the 19th day of embryonic development at which time the precursor frequency was 1/10th that found in the adult thymus. As in the adult thymus, all precursors of TCGF-producing cells had the GK-1.5-positive, Lyt-2-negative phenotype. In parallel to these functional studies, the ontogeny of GK-1.5+, Lyt-2- cells was investigated. In the adult thymus, 80% of cells expressed both GK-1.5 and Lyt-2 antigens, whereas minor subpopulations of 10% and 5% (corresponding to phenotypically mature thymocytes as defined by cortisone-resistant thymocytes [CRT]) expressed GK-1.5 or Lyt-2 exclusively; 3% of cells expressed neither antigen. During ontogeny, thymocytes expressing both GK-1.5 and Lyt-2 first appeared on the 16th day of embryonic development and their proportion increased rapidly thereafter. Interestingly, the GK-1.5+, Lyt-2- subpopulation first appeared in significant numbers on day 19 in parallel with the appearance of functional TCGF activity. Taken together with our previous studies correlating cytolytic T lymphocyte precursor (CTL-P) activity with the Lyt-2+, GK-1.5- subpopulation, these results further emphasize the strict correlation between functional activity and mature surface phenotype of both embryonic and adult thymocytes.