Restriction fragments of fastidious human adenovirus type 41 (Ad41) were cloned in vector plasmid pBR322. A rapid and sensitive nonradioactive molecular-hybridization technique (M. Renz and C. Kurz, Nucleic Acids Res. 12:3435-3444, 1984) showed that one clone specifically detected fastidious Ad40 and Ad41 (subgenus F) without cross-hybridization with nonfastidious adenoviruses. This clone was mapped in a region of the Ad41 genome corresponding to early transcription unit E1B of Ad2. A number of DNAs from fastidious and nonfastidious adenoviruses were extracted, without cultivation, from stools of children with gastroenteritis and were hybridized with an Ad2 probe and with the cloned probe, allowing the differentiation of the two groups of viruses. This method could detect DNA quantities as low as 10 pg and should be particularly suitable for stool samples containing adenoviral DNA in amounts too low to be detected by staining with ethidium bromide.