To determine whether an outbreak of hepatitis A that occurred in 52 Italian hemophiliacs was acquired through the infusion of contaminated factor VIII or through environmental enteric transmission, a case-control study of the first 29 infected patients was carried out. Case patients were neither more nor less likely than controls (hemophiliacs without HAV infection) to have traveled to high-risk countries, consumed raw shellfish or had contact with persons with jaundice. The case patients, however, were more likely than controls to have received a factor VIII concentrate treated with solvent-detergent and to have had larger infusions of the concentrate during the presumed HAV incubation period. In the PCR analysis of HAV sequences from implicated lots of factor VIII, HAV sequences were found in 5 of 12 lots of factor VIII implicated in the outbreak. Three different strains of HAV were recovered, suggesting that lots were not contaminated from the same plasmapheresis donor. To obtain molecular evidence that the HAV detected in the factor VIII preparations was responsible for transmission of HAV, serial serum samples from two patients were tested for HAV gene sequences and compared with those of the implicated lots. The genomic sequences of HAV obtained for two matched sets of factor VIII and recipient serum samples were identical within each set, but different for the two sets.