We assessed the feasibility of using dried serum spots (DSS) for the serological and molecular diagnosis of hepatitis A virus (HAV) infection. Sixty-eight sera spotted onto filter papers (Whatman International Ltd., United Kingdom) were used for detection of total anti-HAV antibodies, and 64 sera were used for detection of immunoglobulin M antibody to HAV. DSS were stored at 4°C, room temperature, and 37°C for 1, 2, and 4 weeks. Sensitivity and specificity of the serological assays were 100% regardless of temperature and storage duration. To assess the stability of HAV RNA, we performed qualitative and quantitative reverse transcriptionPCRs (RT-PCRs) with human plasma spiked with serial dilutions of cultured HAV spotted on Flinders Technology Associates filter paper cards (Whatman International Ltd.). Filter papers were stored at room temperature and processed for RT-PCR assays. No reduction of viral load was observed after 5, 15, and 30 days of storage. The ϳ10-fold reduction of sensitivity from DSS was attributable to a smaller sample input in DSS samples. This method was further evaluated using 35 frozen sera. HAV RNA amplification showed 100% specificity and 92.3% sensitivity, and sequence analysis from DSS and sera provided identical results. HAV RNA can be accurately recovered from DSS for molecular epidemiology purposes, and we confirm the reliability of blotted samples in the serological diagnosis of HAV infection. The DSS method facilitates storage and shipment of samples from routine laboratories to reference centers for further investigations and large epidemiological studies.Hepatitis A virus (HAV) is a major cause of viral hepatitis worldwide. Infection is usually asymptomatic or anicteric in children younger than 6 years of age but can lead to fulminant hepatitis, particularly in adults older than 50 years of age. With the improvement in hygiene conditions in developed countries, there has been a striking reduction of HAV endemicity over the past few decades (13,25). This decrease in natural immunization allows potentially massive outbreaks involving adults with a consequent number of severe clinical forms and an important socioeconomic impact.The diagnosis of acute hepatitis A is based on the detection of the immunoglobulin M (IgM) antibody to HAV (HAVM). Determination of the total anti-HAV antibodies (HAVT) or anti-HAV IgG allows the determination of the HAV immune status. Investigation of outbreaks relies on epidemiological and serological studies, but only molecular investigation is able to link apparently sporadic cases or apparently distinct outbreaks (6,24). This methodology requires costly devices, such as thermal cyclers and sequencers, and expertise in nucleotide sequence analysis that are available in only few reference centers. Many studies have demonstrated the good performances of blotted blood, serum, or plasma samples in serological and molecular diagnosis of different viral infections, including infection with cytomegalovirus (23), human immunodeficiency virus (HIV) (3, 18, 19), he...