The nucleotide sequence of the B19-Wi isolate of human parvovirus was determined and compared throughout the open reading frames and putative transcription signals with the sequence of the closely related B19-Au isolate. In vitro run off transcription assays, using B19-Wi DNA as the template, indicated that there is a strong promoter between m.u. 5 and 7. Deletion clones show that a region between nt 258 and 321 is necessary for in vitro transcriptional activity. Primer extension studies identified the start site at 31-32 nucleotides downstream of the sequence TATATATA. The strength of this left-hand promoter is unusual among parvovirus promoters characterized to date, and the possibility of an upstream enhancer element is discussed.