Virus-like particles (VLPs) mimicking the simian-human immunodeficiency virus SHIV89.6P (VLPSHIV) were produced by co-infection of Vero cells with fowlpox SIVgag/pol (FPgag/polSIV) and fowlpox HIV-1env89.6P (FPenv89.6P) recombinant viruses. As a necessary prerequisite for a more efficient vaccine approach, ultrastructural, functional and molecular characterizations of VLP(SHIV) were performed in the SHIV-macaque model to verify the similarity of these particles to SHIV89.6P. Here we show that VLPSHIV can infect T cells by fusion without replication, as demonstrated by the absence of new viral progeny in VLPSHIV-infected C8166 cells. Biochemical characterization showed identical protein profiles of VLPSHIV and SHIV89.6P, and ultrastructural analysis of Vero cells releasing VLPSHIV also confirmed the morphological similarity of these pseudovirions to SHIV89.6P particles. Viral mRNAs were also found packaged inside the core of VLPSHIV by RT-PCR and reverse transcriptase assays.