The nonspecific clinical presentation of acute hepatitis A (AHA) mandates the detection of anti-hepatitis A virus IgM antibodies (IgM anti-HAV) in the serum for obtaining a definitive diagnosis. However, IgM anti-HAV might not be present during the early phase of the disease. The aim of this study was to determine the optimal time for repeating the IgM anti-HAV test (HAV test) in AHA patients with a negative initial test. In total, 261 patients hospitalized with AHA were enrolled for this retrospective study. AHA was diagnosed when the test for IgM anti-HAV was positive and the serum alanine aminotransferase (ALT) level was ≥400 IU/L. Repeat HAV test was conducted after 1-2 weeks if the initial HAV test was negative but AHA was still clinically suspected. The results of the initial HAV test were negative in 28 (10.7%) patients. The intervals from symptom onset to the initial-HAV-test day and from the peak-ALT day to the initial-HAV-test day were significantly shorter in the negative-initial-HAV-test group, but on multivariate analysis only the latter was significantly associated with negative results for the initial HAV test (β=-0.978; odds ratio [95% confidence interval]=0.376 [0.189-0.747]; P=0.005). The HAV test was positive in all patients when it was performed at least 2 days after the peak-ALT day. The results of HAV tests were significantly associated with the interval from the peak-ALT day to the HAV-test day. The optimal time for repeating the HAV test in clinically suspicious AHA patients with a negative initial HAV test appears to be at least 2 days after the peak-ALT day.