Diagnosis of acute hepatitis A virus (HAV) infection is based on the detection of HAV immunoglobulin M (IgM). However, IgM could be detected due to nonspecific polyclonal activation of the immune system. An avidity test for anti-HAV IgG was developed to distinguish acute infection, where low-avidity antibodies are detected, from immune reactivation. The assay was tested on 104 samples, including 11 sera from patients with past infection, 15 sera from patients with acute infection and 4 collected after recovery, 10 sera from vaccinated subjects, 4 sera from patients with suspected immune reactivation, and 60 unselected HAV-IgM positive sera, collected over 1 year in a routine laboratory. The avidity index (AI) was expressed as percentage. The results were provided as the mean +/- one standard deviation. Patients with a history of prior infection had AIs of >70% (mean, 86% +/- 10), whereas the mean AI was 36% +/- 16 during acute HAV infection (P < 0.001). Within the first month after the onset of hepatitis, avidity was either noncalculable due to a very low IgG titer or <50%. In patients with immune reactivation, avidity was >70% (88% +/- 10%), a finding consistent with a prior infection. Among the 60 unselected sera, 35 (58%) had a noncalculable or <50% avidity, and most of them had a detectable HAV RNA, confirming HAV infection. In contrast, 16 (27%) had an avidity of >70%, and none was reverse transcription-PCR positive, suggesting immune reactivation. These 16 patients were significantly older than the others (50 +/- 16 years versus 26 +/- 14 years). The new anti-HAV IgG avidity assay we developed could improve HAV infection diagnosis, particularly in elderly patients.