Retroviral vectors have been developed for gene therapy of blood disorders because they achieve long-term expression of the transgene. However, interactions between the regulatory elements contained in such vectors and cellular genes may result in pathogenic proto-oncogene activation. We designed a cassette consisting of a splice acceptor followed by the coding sequences for Green Fluorescent Protein (GFP) and a polyadenylation site which was inserted in a reverse orientation in self-inactivating lentiviral vectors. Retroviral vectors integrate preferentially in genes and in one-half of the integrations, the expression cassette will be in a tandem orientation with respect to the gene's promoter and potentially expressed when the trapped promoter is intrinsically active or activated by regulatory elements within the vector. Approximately 10% of the integrations of the control vector with only the GFP cassette resulted in expression of the GFP marker. The trapping cassette in vectors containing globin regulatory elements was expressed more frequently in erythroleukemia (K562) cells than the control cassette only vector but there was no increase in promoter trapping efficiency in HeLa cells. In contrast, we found that addition of a gammaretroviral long terminal repeat increased the frequency of GFP expression both in K562 and HeLa cells. Promoter activation in K562 cells by vectors containing globin regulatory elements was significantly reduced by addition of flanking insulator elements.