Genetic and antigenic characterizations of 70 strains of adenovirus type 41 (Ad41), isolated between 1998 and 2001 from children in Japan, Vietnam, and Korea, were done by DNA restriction enzyme (RE) analysis, sequencing analysis, and monoclonal antibody (MAb)-based enzyme-linked immunosorbent assay (ELISA). Eight genome types were observed in the present study, among which D25, D26, D27, and D28 were novel genome types. These eight genome types were divided into two genome-type clusters (GTCs) based on phylogenetic analysis of the hypervariable regions (HVRs) of the hexon. GTC1 includes D1, D25, D26, D27, and D28, and the GTC2 contains D4, D12, and D22. The amino acid homologies among the members within a GTC were 97 to 100%, whereas between the members of different GTCs the homologies were 92 to 94%. The specificity of the GTC classification was confirmed by ELISA with MAb 1F, which was selected by the Ad41 prototype Tak strain. It was found that only the isolates of GTC1 but not of GTC2 reacted with MAb 1F. These results suggest that Ad41 isolates from the three countries should be classified into two subtypes. The accumulation of amino acid mutations located in HVRs of hexon are indicative for the classification of Ad41 subtype.Adenoviruses are responsible for a wide range of disease symptoms. To date, there are 51 recognized serotypes of human adenovirus that are classified into six subgenera (A to F) based on several antigenic, morphological and molecular criteria. Among them, subgenus F, represented by the two serotypes adenovirus type 40 (Ad40) and Ad41, is associated with diarrhea in children; it is found in 1 to 20% of fecal specimens from children with acute gastroenteritis (3,9,12,15,22,35). The two serotypes (Ad40 and Ad41) of subgenus F are termed enteric adenovirus (EAd) because of their tropism for the gastrointestinal tract (10, 32). The subgenus F adenoviruses grow poorly in most cell culture systems, in contrast to other cultivable "nonenteric" adenoviruses, and are therefore also termed "noncultivable" or "fastidious" adenovirus (7, 16).Earlier surveys had shown that the occurrences of Ad40 and Ad41 are approximately equal (6, 11). However, several studies have recently reported a decrease in the rate of isolation of Ad40 and a concomitant increase in the rate of isolation of Ad41 (5,8,25,34). After 1986, Ad41 infection became dominant over Ad40. Our previous studies confirmed that Ad41 was the prevailing serotype of adenovirus associated with acute diarrhea among children also in Japan, Vietnam, and Korea (19). The change in prevalence of Ad41 might have been caused by an antigenic drift, thus increasing the incidence of infection in susceptible individuals (3). Therefore, it is important to characterize these Ad41 strains. In the last decade, there were relatively few comprehensive epidemiological studies of subgenus F adenoviruses from children with diarrhea in Asian countries. The objectives of the present study were (i) to describe the genome types of Ad41 isolates by DNA restriction end...