An adenoviral vector carrying a 2-Kb fragment of the K-ras proto-oncogene inserted in antisense orientation with respect to the cytomegalovirus promoter was constructed and used to infect H460a lung cancer cells (codon 61 K-ras mutation). The gene was efficiently transferred, and a high level of expression of antisense K-ras was achieved. At a multiplicity of infection to achieve 65% transduction of cells, the expression of K-ras protein was reduced by 70% in the lung cancer cell line H460a as compared with cells infected with control vectors or noninfected cells. This reduction produced a 47% inhibition of monolayer growth and a 90% inhibition of colony formation. At a similar level of transduction in the cell line H358 (codon 12 K-ras mutation), a 59% inhibition of monolayer growth compared with control vectors occurred; however the inhibition of H322 cells (wild-type k-ras) growth was no different than control vector infected cells. These data suggest that the adenoviral K-ras H322a antisense vector may have therapeutic potential in tumors in which K-ras is mutated.