Spleen necrosis virus (SNV) is an avian retrovirus that can infect some mammalian cells such as dog cells as well as all avian cells tested to date. We were interested in testing whether SNV could also infect primate cells. For these experiments, we used HeLa and COS-7 cells. Initially, we determined whether the SNV long terminal repeat promoter was functional in HeLa and COS-7 cells. In transient transfection assays, the SNV promoter efficiently directed chloramphenicol acetyltransferase gene expression in both HeLa and COS-7 cells. Using SNV- and murine leukemia virus-derived retroviral vectors containing the neomycin phosphotransferase gene, we found that SNV established a provirus in HeLa and COS-7 cells as efficiently as did an amphotropic murine leukemia virus, as judged by the number of G418-resistant HeLa and COS-7 cell colonies obtained after infection and selection. Although SNV formed a provirus in both HeLa and COS-7 cells, productive infection of these cells was not obtained with use of replication-competent SNV. These results suggest that SNV can infect, form a provirus, and stably express a transduced gene in primate cells, but there is a posttranscriptional block to its replication in these cells.