Most short hairpin RNA (shRNA)-expressing vectors use RNA polymerase III promoters, as expression of shRNAs from RNA polymerase II promoters is not well understood, due to the lack of defined transcription initiation sites and functional localization of the transcription termination signal. Here we describe a modified cytomegavirus (CMV) promoter, an RNA polymerase II promoter, to express shRNAs with only four overhangs at the 5' end. The expression of shRNAs from the modified CMV promoter was terminated by the transcription termination, the polyadenylation, or the poly(U) termination signal. These results demonstrated that the poly(U) and polyadenylation termination signals are interchangeable for terminating the expression of shRNAs from the CMV promoter and result in similar efficacies in inhibiting endogenous target genes in mammalian cells. (C) 2004 Elsevier Inc. All rights reserved.