Superantigen assays are used to determine the stimulation of classes of T cells in response to the presence of a superantigen. This requires that antigen-presenting cells, such as B-lymphocytes, are co-cultured with syngeneic T cells over a four-day period. This prerequisite rules out conventional transient transfection procedures, since the cells must remain metabolically active for around five days. Consequently, stably transfected B cells have previously been used to test recombinant DNA constructs for superantigen activity. Here we present a protocol for the transient transfection of B cells that results in metabolically viable cells. Mouse mammary tumor virus (MMTV) encodes an endogenous superantigen. Using this transient transfection procedure, we show that a number of MMTV-based constructs give rise to functional superantigen activity. The ability to transiently transfect lymphocytes and maintain their viability should greatly facilitate studies of genes encoding products, such as superantigens, that can only be indirectly assayed on a second cell type as a result of expression in the recipient cell.