The regulation of TT virus (TTV) gene expression was characterized. Transient-transfection assays using reporter constructs revealed that a 113-nucleotide (nt) sequence within the untranslated region, proximal to the transcription initiation site and containing a TATA box motif, has a basal promoter activity. This sequence is well conserved among different TTV genotypes. Upstream stimulating factor bound to a consensus binding motif within this region and positively regulates TTV transcription. Furthermore, a 488-nt region upstream of the basal promoter exhibited enhancer activity, presumably in a cell type-specific manner. This study illustrates some of the mechanisms involved in the transcriptional regulation of TTV.