A novel helper-free adenovirus type 5 (Ad5) vector system, which utilizes a cloning site 0.2 kilobase (kb) from the right end of the genome, has been developed. To construct a nondefective Ad5 bearing the 2.8-kb DNA fragment of hepatitis B virus (HBV) at this site, we deleted the 2.1-kb nonessential E3 fragment from cloned DNA covering the right one-fourth of the Ad5 genome (76 to 100 map units), inserted the HBV DNA into this site, ligated the recombinant DNA to the rest of the Ad5 genome, and transfected the ligated DNA into human embryo kidney cells. Most of the recovered virus clones had only the E3 deletion and no HBV insertion, suggesting that a homologous recombination occurs between transfected DNAs in these cells. The isolated Ad5 virus bearing the HBV DNA (Ad5-HBL) grew without helper virus in HeLa cells as efficiently as wild-type Ad5, although the 1.9-kb major E4 transcript was detected only poorly in the early phase in the Ad5-HBL-infected cells, suggesting that the HBV DNA inserted upstream of the E4 promoter reduces the E4 transcript. HBV mRNAs transcribed from the inserted DNA were at least as abundant as Ad5 early mRNAs in the late phase of Ad5-HBL infection, but the HBV surface antigen was barely detectable in the infected-cell lysate and culture medium. This result suggests that HBV mRNAs can be transcribed from the inserted genes but no protein can be translated from the HBV mRNAs, presumably because of the translational suppression of cellular mRNAs caused by adenovirus in its late phase.