The Adenoviridae family consists of five genera, of which the genus Mastadenovirus includes human viruses classified into 57 serotypes clustered into 7 subgroups (A-G). Serotypes 40 and 41 (subgroup F) are specifically associated with childhood gastroenteritis and are the most common cause of acute gastroenteritis in young children after rotaviruses and noroviruses. Standard methods for laboratory diagnosis of adenovirus infection include electron microscopy (EM) and conventional cell culture (CCC), although it is widely considered that adenoviruses 40 and 41 are difficult to cultivate (Ko et al., 2003; Kim et al., 2010; Sharma Dey et al., 2011), such that their circulation is most likely underestimated. One hundred and ten faecal specimens from paediatric patients with gastroenteritis were confirmed positive for adenovirus by EM and/or CCC at the Virology Unit of the University-Hospital of Parma (Italy) during the period January 2010-December 2012. They were analysed to determine the actual prevalence of adenovirus 40 and 41 in these patients using PCR and restriction endonuclease analysis, and to evaluate their cultivability in standard cell lines. The results showed a high prevalence of subgroup F (62.7%), among which serotype 41 (89.8%) predominated on serotype 40 (10.2%). Surprisingly, among the 75 adenoviruses isolated by CCC, 37 (49%) belonged to subgroup F, suggesting a higher capacity of adenovirus 40 and 41 to replicate in cell culture than previously thought. PCR and restriction enzyme techniques provide an efficient means of diagnosing enteric adenoviruses correctly, including subgroup F adenovirus strains in young children with gastroenteritis.