The anterior chamber of the eye is an immunosuppressive microenvironment as shown experimentally by immune privilege, anterior chamber-associated immune deviation, and inability to display local delayed-type hypersensitivity responses. It recently was reported that both the aqueous humor and the cells of the iris and ciliary body (I-CB) have immune inhibitory properties in vitro, suggesting that these components of the anterior segment might contribute to the unique properties of this microenvironment. To explore the cellular sources of immunosuppressive factors in the anterior chamber, cultures of I-CB cells were established from normal eyes of BALB/c mice. Supernatants were harvested from these cultures and assayed in vitro for their ability to inhibit T-lymphocyte activation. It was found that I-CB cell-derived supernatants profoundly suppressed alloantigen-driven T-cell proliferation (mixed lymphocyte response) and interleukin-2 production by a T-cell hybridoma that responds to stimulator cells bearing I-Ad. The inhibitory activity of I-CB supernatants did not appear to be related to prostaglandins; supernatants of I-CB cells cultured with indomethacin retained their suppressive properties, as did supernatants to which neutralizing antiprostaglandin E2 antibodies had been added. Moreover, suppression by I-CB supernatants was not relieved by antibodies specific for transforming growth factor-beta, even though this cytokine is known to be present in normal aqueous humor. Thus, the identity of the suppressive factor(s) in cultured I-CB cell supernatants remains elusive. Finally, by separating I-CB cell suspensions into bone marrow-derived (T-200-positive) and those not derived from bone marrow (parenchymal) subpopulations with a fluorescence-activated cell sorter, it was determined that the inhibitory activity of I-CB cell suspensions was produced by parenchymal, rather than hematogenous, cells. It is proposed and discussed that inhibitory factors and cytokines secreted by parenchymal I-CB cells contribute to the immunosuppressive qualities of the anterior chamber.