A yellow fever virus of a South American genotype was identified in the liver and blood samples of a non-vaccinated European patient after his return from Brazil. ELISA tests were negative for IgG and positive for IgM against yellow fever. Yellow fever proteins in the formalin-fixed and paraffin-embedded liver biopsy were detected by immunohistochemical procedures. Viral RNA extracted from the liver tissue was also detected using an RT-semi-nested PCR procedure and molecular hybridization. Alignment of the sequence obtained from a gene fragment amplified by RT-semi-nested PCR directly from a blood sample with those of African and South American yellow fever virus strains identified a Brazilian topotype as being responsible for the disease. RT-semi-nested PCR may be used advantageously for clinical specimens for rapid and specific diagnosis, and with archival biopsy material for retrospective studies.