Hepatitis A virus (HAV) isolates from around the world have been classified into seven genotypes (I-VII). Most human strains belong to genotype I, which has been divided into two subgenotypes, A and B. South America has provided a small number of strains studied at the genome level. In the present study, IgM anti-HAV antibodies were detected in 116 out of 250 (46%) serum samples collected from consecutive patients with acute hepatitis referred to the Brazilian Reference Center for Viral Hepatitis, Rio de Janeiro. Viral RNA were extracted from all 250 samples and submitted to a reverse transcription-polymerase chain reaction (RT-PCR) assay designed to amplify a genome segment in the VP1/2A junction region. HAV RNA was detected in 54/116 (47%) and 17/134 (13%) IgM anti-HAV-positive and -negative sera, respectively. In addition, HAV RNA was detected in 17/35 (49%) IgM anti-HAV-positive sera that had been collected at a day care center where cases of acute hepatitis were being observed for 3 months. Nucleotide sequences (168 bp) of PCR products were determined for 30 HAV isolates. Phylogenetic analysis showed that 21 belonged to subgenotype IB, while 9 were of subgenotype IA. Interestingly, a concomitant circulation of isolates from subgenotypes IA and IB was observed in the day care center.