First-generation adenoviral vectors induce G(2)/M arrest and cell death at high multiplicities of infection (m.o.i.'s) in vitro. It is unclear whether this cytotoxicity is entirely adenoviral gene related or influenced in part by the encoded transgene. We examined this question in epithelial cells using seven vectors at relatively low (50) or higher (200) m.o.i.'s. The vectors contained no transgene (+/-promoter), transgenes encoding a cytoplasmic reporter protein (two luciferase constructs; P-galactosidase), or transgenes encoding a secretory protein (alpha 1-antitrypsin; growth hormone). After 24 h with a m.o.i. of 50, vectors encoding cytoplasmic reporter proteins led to greatest cytotoxicity (similar to 35-40% cells in G(2)/M). Vectors without a transgene resulted in lower cytotoxicity (similar to 15%, minus, or 23%, plus promoter, cells in G(2)/M). Vectors encoding secretory proteins led to similar to 22-25% cells in G(2)/RI. A similar pattern resulted when cell number was measured. Results were unrelated to the steady-state levels of transgene product. At the higher m.o.i., all vectors caused substantial growth retardation, This is the first demonstration that adenoviral vector-induced cytotoxic effects are in part related to the transgene encoded.