The mechanism of the action of antigen specific suppressor cells was investigated in in vitro systems. The main areas under investigation were the target of suppression by secreted suppressor factor (SF), which was found to be an Ly-1 plus activated T cell, presumably a helper cell. The genetic restrictions on SF action were investigated: both responder and non-responder strains made SF, and were suppressed by it, in contrast to previous results by others using extracts of suppressor cells. The nature of suppressor factors was investigated using immunoadsorbents: they have an antigen combining site, I-region controlled determinants (I-J) and a MW of 50-60,000 daltons. In order to produce large quantities of homogenous suppressor factor, antigen specific suppressor cells were hybridized with a thymoma cell line. Some of the hybrids obtained secrete antigen specific SF, which have the same properties as SF secreted by conventional suppressor cells. These hybrids will be analyzed further, and the SF they produce used for biochemical characterization. To further characterize suppressor factors, antisera were raised in rabbits and mice using SF eluted from antigen columns as an antigen. These antisera defined a "constant" and a "variable" region of SF, and permit the analysis whether these regions are H-2 or Ig linked. Attempts will be made to produce SF from human peripheral blood leucocytes.