Objective To engineer recombinant strains of simian-human immunodeficiency virus ( SHIV) stably expressing green fluorescent protein ( EGFP) . A replication-competent SHIV construct containing the green fluorescent gene with the ability to infect rhesus monkeys would serve as an important tool in AIDS research. Methods A SHIV strain was constructed by inserting the EGFP genes into the nef gene of SHIV KB9. The infection activity and bright fluorescence expression of the SHIV clone was determined in vitro in TZM-bl cells and macaque PBMCs. Results Replicationcompetent virus and bright fluorescence of infected cells were obtained with one construct，in which EGFP was inserted into the SHIV nef locus. This strain was infectious to rhesus PBMC and TZM-bl cells. Green fluorescing cells were detected by direct microscopic visualization. Conclusions A recombinant and replication-competent SHIV strain expressing EGFP is engineered. It is suggested that the SHIV-KB9nefGFP could be used as a tool to directly detect infected cells and aid in the immunophenotypic characterization of these cells.