To explore the potential of an adenoviral antisense RNA transcript for gene therapy of cervical cancer, we introduced the antisense RNA transcript of E6 and E7 genes of human papillomavirus (HPV) 16 into cervical cancer cells harboring HPV 16 via a recombinant adenoviral vector, Ad5CMV-HPV 16 AS and analyzed the effects of expression of these genes on cell growth and tumor growth. Ad5CMV-HPV 16 AS contains the cytomegalovirus-promoter, E6 and E7 genes of HPV 16 in antisense orientation, and the SV40 polyadenylation signal in a mini-gene cassette, which is inserted into the E1-deleted region of modified adenovirus 5. The entire E6/E7 region of HPV 16 was amplified by polymerase chain reaction (PCR) before cloning into the mini-gene cassette. By reverse transcriptase-PCR, HPV 16 E6/E7 antisense RNA was detected in SiHa cells infected with Ad5CMV-HPV 16 AS. The growth of the Ad5CMV-HPV 16 AS-infected cells was greatly suppressed, as evidenced by a decrease in cell count. The growth inhibitory effect of Ad5CMV-HPV 16 AS was significantly enhanced by an adenoviral p53 construct, Ad5CMV-p53. In an ex vivo study in nude mice, tumorigenicity was completely inhibited in mice injected with Ad5CMV-HPV 16 AS-infected SiHa cells. These data suggest that transfection of cervical cancer cells with HPV 16 E6/E7 antisense RNA in a form such as Ad5CMV-HPV 16 AS is a potential novel approach to the therapy of HPV 16-positive cervical cancer.