Direct inoculation of genetic material in DNA form is a novel approach to vaccination that has proved efficacious for a number of viral agents. We are interested in the potential of this approach for the delivery of vaccines based on attenuated or replication-defective retroviruses. Toward this goal, we tested the effect of intramuscular inoculation of a plasmid containing the entire genome of feline immunodeficiency virus (FIV-Petaluma, F14 clone). DNA delivery was compared with intramuscular or intraperitoneal inoculation of virus reconstituted from the same molecular clone. The outcome was monitored by serological analysis and quantitative virus load determination over a 31-week period. DNA inoculation was found to be a reliable means of infection, although seroconversion and the rise in PBMC virus load were delayed relative to intramuscular or intraperitoneal inoculation of virus. At 31 weeks, similar levels of proviral DNA were detected in central lymphoid tissue of all infected animals. In conclusion, DNA inoculation of proviral DNA will be of use as a novel method of cell-free virus challenge and may have further potential for the delivery of lentiviral vaccines.