Oncoretrovirus, but not lentivirus, displays a high transcriptional readthrough activity in the 3' long terminal repeat (LTR) (Zaiss et al. J. Virol. 76, 7209–7219, 2002). However, the U3-deleted, self-inactivating (SIN) lentiviral LTR also exhibits high transcriptional readthrough activity. Since the canonical "core" polyadenylation signal (AAUAAA) of the lentivirus is located in the R-U5 region, the above finding suggests that additional RNA termination signals must be present in the U3 region. Insertion of alternative termination signals including panhuman T cell leukemia virus type I polyadenylation signal, a 3' end small intron, and a tertiary tRNA motif into the lentiviral SIN LTR did not restore the transcriptional termination function. Functional dissection of the U3 region revealed that 70–80% of the termination signals reside in the transcriptional control region within 124 nt overlapping NFκB, Sp1 and TATA binding sites. Serial deletion analysis of the transcriptional control region indicates that the lentiviral enhancer/promoter elements are essential to the RNA termination function. These results characterize the mechanism of lentiviral transcriptional readthrough, which addresses important fundamental and practical issue of RNA readthrough influencing lentiviral gene function and vector safety.