Equine infectious anemia virus (EIAV) is a member of the lentivirus subfamily of retroviruses. Lentiviruses replicate in cells of the immune system, and activation of immune cells has been shown to modulate virus replication. Studies were undertaken to determine the effects of macrophage activation on EIAV replication in vitro. Primary horse macrophage cultures (HMC) were established from 20 different horses, infected with EIAV, and stimulated with 5 [mu]g/ml of bacterial endotoxin. Supernatants collected from HMC were assayed for the presence of tumor necrosis factor-[alpha] (TNF-[alpha]), and titers of infectious virus. Results indicated that EIAV replication in vitro varied significantly (p ≤ 0.0001) from horse to horse, regardless of the treatment of HMC. Also, EIAV replication was significantly (p ≤ 0.0001) decreased in HMC stimulated with bacterial endotoxin as compared to untreated HMC. No significant correlation was found between virus replication and production of TNF-[alpha] following endotoxin treatment of virus-infected cells. However, inhibition of EIAV replication was more dramatic when basal levels of TNF-[alpha] were detected at the time of infection, suggesting that cellular factors present in activated macrophages may inhibit EIAV replication early after infection. Additional experiments were designed to determine the mechanism of inhibition of EIAV replication in activated HMC. HMC were treated with serially diluted bacterial endotoxin and infected with EIAV. Total cellular DNA and RNA were isolated from HMC at six hours, 18 hours, 43 hours and six days following infection, and the polymerase chain reaction (PCR) and the reverse transcription-PCR were used to amplify EIAV proviral DNA and mRNA sequences, respectively. Results indicated that the reverse transcription of proviral DNA early after infection was not affected by endotoxin treatment of HMC. Also, there were no quantitative differences in full-length or multiply-spliced mRNAs until six days post-infection. However, variable splicing patterns dependent on both time and dose of endotoxin were observed in mRNA species from endotoxin-treated HMC. The appearance of single-spliced EIAV mRNA was delayed by endotoxin treatment. Together these results suggest that inhibition of EIAV replication in endotoxin-treated HMC occurs during the transition from early to late stages in virus replication.