We have previously reported complex effects of cytokine-containing T cell supernatants on the interleukin (IL)4 plus phorbol 12-myristate 13-acetate (PMA)-induced proliferative response of murine thymocytes. Here we show that recombinant murine IL-2, IL-6, and IFN-gamma each differentially regulate the IL-4/PMA-driven growth of thymocyte subpopulations. Thymocytes fractionated into four subpopulations on the basis of CD4 and CD8 expression were stimulated to proliferate by IL-4/PMA. Interferon-gamma (IFN-gamma) caused almost complete inhibition of the CD4+/CD8- response but had no measurable effect on the growth of CD4-/CD8+ or CD4-/CD8- populations. This inhibitory effect was also observed on splenic CD4+/CD8- T cells. In contrast, IL-6 strongly enhanced the proliferative response of CD4+/CD8- thymocytes, but showed no effect on peripheral CD4+/CD8- T cells, suggesting that IL-6 may be an important regulator of growth in the thymus. IL-2 also enhanced the proliferation of both CD4-/CD8+ and CD4-/CD8- thymocytes to IL-4 and PMA. To test whether the IL-4/PMA stimulus provided all the signals required to initiate growth in each subpopulation, we titrated cell number and examined the relationship between cell dose and cell response. Growth of CD8+/CD4- cells was cell density independent, indicating that IL-4/PMA is sufficient stimulus to induce growth of these cells. In contrast, growth of CD4-/CD8- and CD4+/CD8- cells is cell density dependent, suggesting a requirement for another signal provided by the cells themselves. These observations suggest that more signals remain to be identified in this thymocyte growth system.