The relatively low efficiency of target cell transduction and variations in the stability of transgene expression by retroviral vectors based on the Moloney murine leukemia virus (MoMLV) are major impediments to the use of such vectors in cancer gene therapy approaches. The present study was designed to investigate the stability and efficiency of transgene expression in human lung and breast cancer cell lines transduced with vectors based on human immunodeficiency virus type 1 (HIV-1) in vitro and in vivo in nude mouse models of metastasis. H460 lung carcinoma cells and MDA-MB-231 breast carcinoma cells were transduced with lentiviral vectors encoding enhanced green fluorescent protein (EGFP) and beta-galactosidase (beta-Gal), respectively. Transduced H460 cells were administered to nude mice by either intravenous or subcutaneous injection and MDA-MB-231 cells were implanted orthotopically into the mammary fat pad of such mice to induce primary tumor and metastatic lung tumor formation. High-level EGFP expression was maintained in transduced H460 cells in metastatic lung nodules for up to 6 weeks and transgene expression in vitro persisted for at least 23 days after retrieval of EGFP-positive H460 cells from the lungs of tumor-bearing mice and subsequent cultivation in vitro. Likewise, beta-Gal expression levels in metastatic MDA-MB-231 cells in lungs remained high for up to 11 weeks. Southern blot analyses carried out with DNA from lung nodules showed that proviral DNAs in H460 cells were maintained stably over many cell generations and during subsequent reimplantation in vivo. However, molecular analyses revealed variations in transgene copy numbers and expression levels among individual lung clones. These results demonstrate the usefulness of HIV-1-based lentiviral vectors for sustained and stable transgene expression in human lung and breast cancer cell lines in vitro and in vivo.