The minor lymphocyte stimulating (MLS) superantigens of mice are encoded by open reading frames (ORFs) in the 3' long terminal repeats (LTRs) of endogenous mouse mammary tumor viruses. By stimulating all T cells bearing particular TCR V beta proteins, these viral superantigens (v-SAGs) exert profound effects on T cell development and function. We have examined expression of the 1.7 kb mRNA product predicted to encode v-SAG proteins in different cells and tissues of the immune system. The LTR-ORF mRNA was expressed in activated B cells and activated mature CD8 but not CD4 T cells, consistent with previous functional studies assessing MLS activity in these cell types. Little or no message was detected in thymic epithelial cells, macrophages, or dendritic cells, although low levels could be observed in thymic epithelium after southern hybridization to PCR-amplified cDNA. LTR-ORF mRNA was also expressed in immature CD4-CD8- and CD4+CD8+ thymocytes, suggesting selective down-regulation of expression in the T cell lineage after differentiation to the CD4+ phenotype. Thus, among cells of the immune system, v-SAG encoding mRNA is expressed predominantly within the lymphoid lineage.