We describe the development of an experimental system to test the hypothesis that the efficiency of retrovirus transduction is dependent on the pathway of virus entry into the host cell and the intracellular trafficking itinerary of the cellular receptor with which it interacts. The experimental system consists of three model target cell lines, derived from HeLa cells, that stably express one of three interleukin-2 receptor a chain (CD25) chimeras, TAC, TAC-CD16, and TAC-DKQTLL, which have identical extracellular domains but different intracellular trafficking itineraries, and a targeted amphotropic murine leukemia retrovirus whose envelope proteins were modified to include a binding site for TAC at their N-termini. We found that the efficiency of retrovirus transduction was affected by the distribution and trafficking itinerary of the TAC receptors. Transduction of cells that expressed TAC-DKQTLL was nearly 4-fold lower than transduction of control cells that did not express any of the TAC receptors. In contrast, transduction of cells that expressed TAC was 1.6-fold higher than transduction of control cells, whereas transduction was not significantly affected by the expression of TAC-CD16. Our results suggest that in the course of designing a targeted retrovirus it may be prudent to target only those receptors that internalize retroviruses via pathways that most efficiently support post-binding steps of infection.