The goal of this work was to determine whether a stable 293 amphotropic packaging line, which we have designated 293-SPA, is useful for the production of high-titer stable virus by comparison to the murine psiCRIP line. Here, we report our unexpected findings that particles derived from the 293-SPA line transduce target cells (both NIH-3T3 cells and primary melanoma cells) with greatly enhanced efficiencies (at least 10-fold) compared to particles derived from the psiCRIP packaging line. We show that the presence of a transferable inhibitor in the psiCRIP line at least partially accounts for this dramatic difference in transduction efficiency. This work has important implications for improving the efficiency of retrovirus-mediated gene transfer in general as well as in the design of new packaging cell lines.