The possible activation of cellular proto-oncogenes as a result of clonal transformation is a potential limitation in a therapeutic approach involving random integration of gene vectors. Given that enhancer promiscuity represents an important mechanism of insertional transformation, we assessed the enhancer activities of various cellular and retroviral promoters in transient transfection assays, and also in a novel experimental system designed to measure the activation of a minigene cassette contained in stably integrating retroviral vectors. Retroviral enhancer–promoters showed a significantly greater potential to activate neighboring promoters than did cellular promoters derived from human genes, elongation factor-1α (EF1α) and phosphoglycerate kinase (PGK). Self-inactivating (SIN) vector design reduced but did not abolish enhancer interactions. Using a recently established cell culture assay that detects insertional transformation by serial replating of primary hematopoietic cells, we found that SIN vectors containing the EF1α promoter greatly decrease the risk of insertional transformation. Despite integration of multiple copies per cell, activation of the crucial proto-oncogene Evi1 was not detectable when using SIN-EF1α vectors. On the basis of several quantitative indicators, the decrease in transforming activity was highly significant (more than tenfold, P < 0.01) when compared with similarly designed vectors containing a retroviral enhancer–promoter with or without a well-characterized genetic insulator core element. In this manner, the insertional biosafety of therapeutic gene vectors can be greatly enhanced and proactively evaluated in sensitive cell-based assays.