In lentiviral gene delivery systems, transgene expression cassettes are commonly cloned without a polyadenylation signal to prevent disruption of full-length lentiviral genomes on mRNA maturation in producer cells. The lack of the polyadenylation signal, however, has the potential to reduce stability and translation efficiency of transgene mRNA. Therefore, we have assessed the effect of a strong internal polyadenylation [poly(A)] signal on both transgene expression levels in virus-infected cells and functional viral titer, in a series of eight self-inactivating lentiviruses expressing the mOrange transgene under the control of the constitutive cytomegalovirus (CMV), elongation factor 1alpha (EF1alpha), and beta-actin promoters or the highly tissue-specific prostate-specific antigen/probasin hybrid (PSA/Pb) promoter with or without a simian virus 40 (SV40) early polyadenylation signal downstream of the mOrange-coding sequence. We show that mOrange expression levels in virus-infected HEK-293, LNCaP, and primary prostate epithelial cells were increased 3- to 6.5-fold when an internal polyadenylation signal was present. When the CMV and EF1alpha promoters were used, functional viral titer decreased 8- to 9-fold in the presence of the polyadenylation signal, but titer was not affected when transgene expression was driven by the beta-actin promoter or tissue-specific PSA/Pb promoter. We therefore conclude that an internal polyadenylation signal in lentiviral vectors has a highly beneficial effect on transgene expression, but reduces viral titer in a promoter-dependent manner.