A retroviral vector system was developed to transduce a K-ras antisense construct efficiently into human cancer cells. A 2-kb fragment of K-ras gene DNA in antisense orientation was linked to a β-actin promoter and inserted into retroviral vector LNSX in two different orientations. The constructs were transfected into amphotropic packaging cell line GP + envAm12 followed by alternating transduction between the ecotropic packaging cell line ψ-2 and GP + envAm12. Titers up to 9.7 × 10⁷ colony-forming units (cfu)/ml were achieved without detectable replication-competent virus. The human large cell lung carcinoma cell line H460a, which has a homozygous codon 61 K-ras mutation, was transduced with an efficiency of 95% after five to seven repeated transductions. DNA polymerase chain reaction (PCR) and genomic DNA Southern blot analysis showed that the retroviral construct was integrated into the genome of H460a cells. K-ras antisense RNA expression was detected in the cells by Northern analysis, slot blot hybridization, and reverse transcriptase-PCR. Translation of the mutated K-ras p21 protein RNA was specifically inhibited, whereas expression of other p21 species was unchanged. Proliferation of H460a cells was suppressed 10-fold following transduction by the antisense construct. Colony formation in soft agarose and tumorigenicity in an orthotopic lung cancer model in nu/nu mice were dramatically reduced in H460a cells expressing antisense K-ras. We conclude that an antisense construct for K-ras can be expressed effectively in a retroviral vector that can efficiently transduce human cancer cells.