The open reading frame contained within the long terminal repeat (LTR) of mouse mammary tumor virus encodes Naf, a negative regulator of transcription, as well as a superantigen activity, Sag, which causes the deletion of specific classes of T cells. In the present study, the effect of Naf expression on different promoters and the coding requirements for Naf and Sag have been investigated. Sag activity was found to require only sequences in the LTR, whereas sequences located within the gag gene were additionally required for functional Naf activity. Surprisingly, both the classic promoter and a recently described promoter located in the LTR can give rise to both functional Naf and Sag. Further analysis of Naf revealed that the downregulatory effect was mediated by sequences located in the LTR and that heterologous promoters were also affected by Naf.