Superantigens are products of bacterial or viral origin which stimulate large numbers of T cells as a consequence of the interaction of particular V beta chains of the T cell receptor with class II major histocompatibility complex (MHC) molecules and superantigen on the stimulating cell. The Minor lymphocyte stimulatory (Mls) antigens, originally discovered as strong lymphocyte stimulatory determinants in vitro and subsequently shown to delete T cells expressing specific V beta chains during development, have recently been shown to be genetically linked to endogenous mouse mammary tumour viruses (MTVs). This stimulation is effectuated by an unidentified product encoded by an open reading frame (orf) present in the 3' long terminal repeat (LTR) of MTVs. Using in vitro translation in the presence of rough microsomal vesicles, we show that (i) the orf of MTV encodes a type II transmembrane glycoprotein (N-terminus intracellular, C-terminus extracytoplasmic), and (ii) a cotranslationally secreted orf protein is not produced. We have also isolated and sequenced several endogenous MTV orfs (MTV-1, MTV-6 and MTV-13) which are involved in the deletion of V beta-bearing T cells; each of these sequences are nearly identical to each other. These observations, together with sequence comparisons of several orf genes, lead to a model of action of viral superantigens.