The specific recognition and selection of the HIV-1 packaging signal psi (Psi) sequence which is mediated by Gag protein is believed to be pivotal for selective viral genomic RNA packaging and has been a basis for the development of HIV-based transgene delivery systems. However, the requirement of the psi sequence has been questioned recently by a report postulating that the psi element is not absolutely required for transgene transduction. Here, we used a four-plasmid transgene delivery system and analyzed the results by HIV p24 antigen assay, MT4 infection assay, HT1080 colony assay, and reverse transcription-PCR (RT-PCR). The results clearly demonstrate that the psi sequence must be present for efficient transgene encapsidation and transduction.