The 441-nucleotide (nt) region (nt 5325 to 5766) around the splice acceptor (SA) site (nt 5491) was found to be necessary for high-level expression of gag-containing unspliced RNA of Moloney murine leukemia virus (M. Oshima, T. Odawara, T. Matano, H. Sakahira, K. Kuchino, A. Iwamoto, and H. Yoshikura, J. Virol. 70:2286-2295, 1996). Detailed genetic dissection of the 441-nt region revealed that the 5'-end 64 nt (nt 5325 to 5389) were necessary for high-level expression of the unspliced RNA when the spliced RNA was not produced, while the 3'-side 301 nt (nt 5466 to 5766) containing the SA site were necessary for producing spliced RNA. When the spliced RNA was produced, the unspliced RNA could be expressed at a high level even when the 5'-end 64 nt were absent. Probably the virus sequence ensuring the splicing could produce an RNA structure able to compensate for the function of the 5'-end 64-nt region responsible for the expression of the unspliced RNA.