Certain human cell populations have remained difficult to infect with human adenovirus (Ad) serotype 5 because of their lack of coxsackievirus B-adenovirus receptor (CAR). Native adenovirus fiber compositions, although diverse, cannot infect all tissue types. Recently, a chimeric Ad5/35 fiber was created, which displays an altered tropism from Ad5. We incorporated this chimeric fiber into a helper-dependent (HD) adenovirus vector system and compared HD to E1-deleted (E1Delta) vectors by transgene expression, cell transduction efficiency, and cytotoxicity. K562 cells were infected approximately 50 times more efficiently with the chimeric Ad5/35 fiber compared with the Ad5 fiber. Short-term transgene expression was sustained longer from HD Ad5/35 than E1Delta Ad5/35 vector after in vitro infection of actively dividing K562 cells. Rapid loss of transgene expression from E1Delta Ad5/35 infection was not due to the loss of vector genomes, as determined by quantitative real-time PCR (QRT-PCR), or cytotoxicity, but rather through a putative silencing mechanism.