Routine laboratory testing for adenovirus (Adv) requires a procedure that is rapid and reliable, especially for samples from children and immunosuppressed patients, when diarrhea may signal the onset of severe gastrointestinal disorders. An improved culture technique for Adv isolation, using centrifugation step of 24-well plates and needing only 48 h incubation, was evaluated for 382 stool samples. This technique was compared with conventional tube cell culture and a commercial enzyme-linked immunosorbent assay (ELISA) kit. Adv was isolated in 36 samples (9.4%) by rapid culture, in 32 (8.4%) by conventional culture, and in 42 samples (11%) using genus-specific ELISA. A total of 30 isolates were found to be Adv positive in both rapid and conventional cultures, and half of the Adv-positive rapid culture isolates were identified as serotypes 40/41 using a type-specific ELISA. The improved culture method considerably reduces incubation time and also offers a slightly enhanced sensitivity to Adv serotypes. Combined with appropriate cell lines adapted to the isolation of enteric adenoviruses, it therefore constitutes a valuable laboratory test particularly useful in the diagnosis of gastroenteritis.