The authors have developed a new series of lentivirus vectors based on human immunodeficiency virus type 1 (HIV-1) that can transduce nondividing cells. They replaced the U3 region of the 5' long terminal repeat (LTR) in the vector constructs with the cytomegalovirus (CMV) promoter, resulting in Tat-independent transcription while maintaining high levels of expression. A self-inactivating (SIN) vector was created by deleting 133 bp in the U3 region of the 5' LTR, including the TATA box and binding sites for transcription factors Sp1 and NF-κB. This deletion inactivates the LTR in the proviruses after reverse transcription and integration, reducing the risk of generating replication-competent virus. The SIN vectors can be generated without significant decreases in titer. In vivo experiments showed that a SIN vector containing the green fluorescent protein gene under the control of the internal CMV promoter transduced neurons as efficiently as a wild-type vector. Interestingly, a wild-type vector without an internal promoter also successfully transduced neurons, indicating that the HIV-1 LTR promoter is transcriptionally active in neurons even without Tat. In the rat eye, the wild-type vector predominantly transduced retinal pigment epithelium and photoreceptor cells, while the SIN vector transduced other retinal cells, including bipolar, Müller, horizontal, and amacrine cells. This suggests that the HIV-1 LTR can negatively influence the internal CMV promoter in some cell types. The SIN HIV vectors are safer for gene therapy and have broader applicability for high-level gene transfer and expression in nondividing cells.The authors have developed a new series of lentivirus vectors based on human immunodeficiency virus type 1 (HIV-1) that can transduce nondividing cells. They replaced the U3 region of the 5' long terminal repeat (LTR) in the vector constructs with the cytomegalovirus (CMV) promoter, resulting in Tat-independent transcription while maintaining high levels of expression. A self-inactivating (SIN) vector was created by deleting 133 bp in the U3 region of the 5' LTR, including the TATA box and binding sites for transcription factors Sp1 and NF-κB. This deletion inactivates the LTR in the proviruses after reverse transcription and integration, reducing the risk of generating replication-competent virus. The SIN vectors can be generated without significant decreases in titer. In vivo experiments showed that a SIN vector containing the green fluorescent protein gene under the control of the internal CMV promoter transduced neurons as efficiently as a wild-type vector. Interestingly, a wild-type vector without an internal promoter also successfully transduced neurons, indicating that the HIV-1 LTR promoter is transcriptionally active in neurons even without Tat. In the rat eye, the wild-type vector predominantly transduced retinal pigment epithelium and photoreceptor cells, while the SIN vector transduced other retinal cells, including bipolar, Müller, horizontal, and amacrine cells. This suggests that the HIV-1 LTR can negatively influence the internal CMV promoter in some cell types. The SIN HIV vectors are safer for gene therapy and have broader applicability for high-level gene transfer and expression in nondividing cells.