Camptothecin (CPT), a topoisomerase I-specific inhibitor, was found in this study to inhibit the replication of equine infectious anemia virus (EIAV) in chronically infected CF2Th cells (designated CF2Th/EIAV). By measuring viral reverse transcriptase activity in the culture medium, we demonstrated that treatment for 1 h with noncytotoxic doses of this drug inhibited production by 32 to 52%, whereas continuous exposure to this drug resulted in an 85 to 92% inhibition. No effect on the viability or growth rate of the cells was detected in any of these treatments. Indirect immunofluorescence analysis of the CPT-treated CF2Th/EIAV cells with anti-p26 capsid protein antibodies showed 60 to 85% reduction in the immunofluorescence-positive cells following drug treatment, and radioimmunoprecipitation analysis of these cells showed a comparable decrease of the pr55(gag) precursor protein. These data suggest that CPT acts as an anti-EIAV agent to block virus replication in the chronically infected cells.