Acute gastroenteritis is a major source of morbidity and mortality among young children in developed and developing countries. Human adenoviruses (HAdVs), and in particular species F, are related to childhood diarrhoea worldwide. This study presents the results obtained during an investigation of HAdVs causing acute gastroenteritis in children hospitalized in Rio de Janeiro, RJ, Brazil, from April 1996 to September 2003, as well as in children with diarrhoea living in the slums of Salvador, BA, Brazil, from October 2001 to September 2003. A total of 3060 stool samples was analysed by an enzyme immunoassay for rotavirus and adenovirus (EIARA) and 61 (2%) were found to be positive. HAdV presented with low prevalence throughout the year, with a slight but not significant increase in incidence in late summer and early autumn. Children up to 2 years of age were the most frequently affected (79% of all positive samples). All positive samples were analysed further by generic and species-specific HAdV PCR protocols, confirming 100% specificity of this rapid and inexpensive EIARA. Species F was the most prevalent (65%), despite the occurrence of species A (12%), C, D and co-infection F/D (5% each) and species B and co-infections F/A, F/C and B/D (2% each). In order to type the species F strains as HAdV-40 or -41, generic PCR and a HinfI restriction digest were performed. HAdV-40 and -41 were found to represent 62% (23/37) and 38% (14/37), respectively. These results demonstrated that a combination of generic and species-specific PCRs is useful and reliable for HAdV species and type identification directly from faecal specimens. The results confirmed the endemism of human adenoviruses, mainly species F, in children as aetiological agents of diarrhoea, although the limited sensitivity of EIARA as a screening method may have underestimated their prevalence.