Abstract DNA-mediated gene transfer techniques have been used to study the effectiveness of a novel construction involving the feline leukemia virus long terminal repeat (FeLV LTR) for expressing the mouse H-2 L d gene in mouse and human cells. In this construction, the transcription initiation (promoter) and termination (polyadenylation) functions of the FeLV LTR have been split by insertion of a promoterless H-2 gene between them. An S1 nuclease assay has been developed that makes it possible to measure accumulated L d RNA against a background of endogenous major histocompatibility antigen RNAs in mouse and human cells. In mouse cells, the H-2 L d gene was expressed at approximately equal levels (measured as accumulated RNA) when driven either by its own promoter or by the FeLV LTR construction. In human cells, expression at the RNA level was highest when driven by the FeLV LTR. We conclude that the FeLV LTR construction is useful for expressing foreign genes in human cells.