In vitro synthesis of an antigen-specific T cell suppressor factor (TsF) has been accomplished by using partially purified poly(A)-containing RNA in a rabbit reticulocyte lysate cell-free translation system. The poly(A)-containing mRNA was isolated from a cloned T cell hybridoma that constitutively produces a TsF specific for the synthetic polypeptide antigen poly-(LGlu60LALa30LTyr10)(GAT). The RNA was fractionated by size and translated in vitro. The 16S RNA fraction stimulated synthesis of a biologically active protein that specifically suppressed both the GAT-specific antibody response by spleen cells in vitro and the proliferation response to GAT by lymph node T cells from GAT-primed mice. Further, the suppressor factor had a binding site for GAT, a determinant encoded by the I subregion of the major histocompatibility complex (MHC), and an apparent M(r) 19,000 estimated by functional assays on protein separated by NaDodSO4/polyacrylamide gel electrophoresis. These results indicate that virtually no posttranslational modifications (other than proteolytic cleavage) are necessary to obtain biologically active TsF. Hence, the presence of carbohydrate or other chemical groups does not contribute to either the serological properties of GAT-TsF or its biological properties.