The utilization of herpes simplex virus (HSV) as a vector for gene delivery to the nervous system or as a live vaccine delivery system is dependent on the construction and characterization of disabled virus mutants which are unable to cause disease. Under certain circumstances, however, replication-defective vectors may carry a potential risk if they can be efficiently complemented by a co-infecting wild-type virus. Stocks of defective vectors should, therefore, be free from replication-competent virus, and helper cell lines should be incapable of generating replication-competent virus by recombination between the vector and the complementary gene. We describe a glycoprotein H-negative (gH-) virus/helper cell line combination which generates helper-free defective virus stocks containing replication-competent virus at a frequency no higher than 1 in 10(9) p.f.u. This virus/helper cell system provides a suitable background for the construction of safe replication-defective gene delivery vectors. In vivo studies demonstrate that gH- virus is unable to initiate disease in mice and establishes latency at low efficiency compared to wild-type HSV. To determine whether gH- virus can be complemented by wild-type virus in vivo, mice were infected with a variety of mixtures of these viruses. Complementation was observed in a minority of animals infected with more than 10(6) p.f.u. of both wild-type and defective virus but the most common observation was that the presence of defective virus suppressed entry of wild-type virus into the nervous system.