Lentiviral vectors are among the most efficient tools for gene delivery into mammalian cells. A major goal of lentiviral gene delivery systems is to develop vectors that can efficiently target specific cell types. In the present work, we attempt to generate viral particles for targeting gene delivery. We have used CCR5-positive cells as the target for our strategy. Therefore, we developed a novel Sindbis pseudotyped lentiviral vector where the Sindbis receptor binding envelope protein was modified to directly encode a single-chain antibody fragment (scFv) against the CCR5 chemokine receptor. We have generated two chimeric scFv-Sindbis envelopes, varying the length of the peptide linker that connects the heavy chain and light chain of anti-CCR5 scFv. The two chimeric scFv-Sindbis envelopes were successfully incorporated into lentiviral-derived vectors, and the resulting pseudotyped viral particles showed specific targeting to CCR5-expressing cells. However, our data demonstrate that the length of the peptide linker significantly affects the efficiency of infection. Pseudotyped viral particles, which display single-chain antibody fragments with longer peptide linkers, allowed higher titers of infection. The present study can be a model strategy for specific gene delivery mediated by lentiviral vectors pseudotyped with Sindbis envelope displaying scFv that recognizes specific cellular surface proteins. Furthermore, this strategy has the potential to become a powerful approach for targeting gene delivery in anti- HIV gene therapy due to the important role of CCR5 expression in disease progression.