Concanavalin-A-stimulated human T lymphocytes from healthy donors and from patients suffering from diverse immune disorders were fractionated into rosette-forming (R) and nonrosette-forming (NR) cells. The separation method is based upon the ability of the lymphocytes to bind autologous erythrocytes and form autorosettes. Long-term cultures of the R and NR subpopulations were established. The activity of the culture supernatants on the T cell proliferation of normal human phytohemagglutinin (PHA)-induced lymphocytes and of a murine, interleukin-2 (IL-2)-dependent cytotoxic T cell line (CTLL) was investigated. Only the R cell line-derived supernatants from almost all patients tested evinced potent suppressor activity, those from healthy donors less so. The suppressive function was demonstrated not to be due to a cytotoxic effect since preincubation of the PHA-induced lymphocytes and CTLL cells with the factor did not diminish their proliferative capacity. Our study indicates the existence of a competitive relationship between the suppressor factor and IL-2. We found that inhibition of the proliferation decreased with the addition of increasing quantities of exogenous IL-2. We also observed that preincubating the CTLL cells with IL-2 prior to exposing them to the suppressive factor precludes inhibition of their proliferation. Phenotypic analysis of the suppressor cell line revealed that they were comprised of a T cell population which included OKT4+ and OKT8+ cells and that 99% of the cells formed autorosettes. Preliminary purification of the suppressive factor was performed by ultrafiltration and maximal suppression was exhibited by the fraction of less than 10,000 daltons. The development of suppressor cell lines from the unique population of autologous rosette-forming cells may be very helpful in studying the immunoregulatory properties of these cells and their suppressor activity.