Little is known about the molecular basis for activation of suppressor T cells. In this report we describe two macrophage cell lines, BAC1.2.SC8 and its variant progeny B26, that differ in their ability to activate suppressor T cells. The SC8 line is derived from a (BALB/c x A.CA)F1 (H-2d/f) mouse and is haploid with respect to I-Ed. It is capable of activating I-Ed-restricted helper T cells as well as poly-(Glu50Tyr50)-specific I-Ed-restricted suppressor cells. The B26 variant can activate H-2d-restricted helper T cells but activates H-2k-restricted suppressor cells. The I-Ed molecules of SC8 and of B26 have identical amino acid sequences. This suggests that suppressor T cells either recognize posttranslational modifications of the I-E molecule or that there is another accessory molecule that helps determine the major histocompatibility complex restriction in the activation of suppressor T cells.