To better understand the basis of cell type specificity of JCV replication, we have analyzed the expression of the viral late promoter in glial cells. Using transient transfection procedures, we show that the late gene expression, like that of the early gene, is restricted to glial cells. However, cotransfection with a plasmid producing the JCV early protein, T-antigen, stimulates expression from the JCV late promoter in both glial and non-glial cells. The SV40-encoded T-antigen acts similarly on transcription of JCV late promoter in both cell types. This transacting effect occurs at the level of RNA synthesis, as measured by the rate of transcription, stability of the message, and translation. These results indicate that basal JCV late promoter activity is restricted to glial cells, whereas in the presence of viral early protein this promoter functions in both glial and non-glial cells.