Defective particles are naturally occurring virus mutants that lack one or more genes required for viral replication. Such viruses may affect positively or negatively the symptoms of the disease. Thus, it is of great interest to measure the role played by defective particles in the process of human immunodeficiency virus (HIV) infection since accumulating evidence indicates that a great proportion of HIV genomes are defective. We used defective particles produced by two stable cellular clones (UHC-8 and UHC-18) to investigate whether they can affect replication of infectious viral particles generated by a human T-cell line transfected with a molecular HIV-1 clone. Progeny virus harvested from UHC-8 cells has no reverse transcriptase and integrase proteins, while UHC-18 has no reverse transcriptase protein. We demonstrate here that coinoculation of a T-lymphoid cell line and of peripheral blood mononuclear cells with defective and infectious particles leads to a dramatic inhibition of virus replication. Defective particles do not interfere with virus production from proviral DNA. Rather, the inhibition of reinfection events seems to be their mechanism of action. This model closely parallels the in vivo conditions and demonstrates that defective particles may limit the spread of infection and progression of the disease by reducing the yield of infectious virus.