Using a pair of congenic strains of mice differing only at the Mls haplotype (Mls locus and closely linked genes), BALB/c (Mlsb) and BALB.D2-Mlsa, we have compared the in vitro proliferative responses of Mlsb lymphocytes to Mlsa antigens presented on either lymph node cells (LNC) or peritoneal adherent cells (PAC). Results showed that Mlsa-PAC are stronger stimulators than Mlsa-LNC, and furthermore, that the supernatant from Mlsa-PAC may be effective in eliciting a lymphocyte proliferative response. The proliferation in response to PAC supernatant is partially due to activation by nonspecific factor(s); however, the response in the presence of Mlsa incompatible PAC supernatant is about three times greater than the response obtained in the presence of syngeneic Mlsb-PAC supernatant, suggesting an additional stimulation by soluble Mlsa antigens. Contrasting with the ability of PAC-supernatant to stimulate a primary proliferative response in vitro, the in vivo immunization of Mlsb mice with Mlsa-PAC supernatant abrogates the specific proliferative response in subsequent one-way mixed lymphocyte cultures. This abrogation of the specific response is comparable to that observed after immunization with intact Mlsa peritoneal or spleen cells, although in the latter case the anti-H-2 proliferative response is also decreased, regardless of whether the H-2 incompatible stimulating cells express an additional incompatibility for Mlsa. The proliferation of untreated, but not of Mlsa-immunized BALB/c LNC, is stronger in cultures with DBA/2 stimulating cells (incompatible for Mlsa and other non-H-2 antigens) than in cultures with BALB.D2-Mlsa cells (incompatible for Mlsa alone), and is comparable in intensity to that activated by H-2 incompatibility. We conclude that Mlsa antigens are more efficiently recognized by unprimed helper T cells when presented on PAC than when presented on LNC. In the primary proliferative response, the effects of Mlsa and other non-H-2 antigens may be cumulative. In vivo immunization against Mlsa antigens results in suppression of the specific proliferative response and, to a certain extent, of the nonspecific proliferative response (directed against both H-2 and other non-H-2 antigens). Since Mlsa antigens are obtainable in soluble form, their physico-chemical purification can now be envisaged.