An outbreak of acute hepatitis of unknown aetiology in children was first reported in Scotland in April 2022.1 Cases aged <16 years have since been identified in 35 countries.2 Here we report a detailed investigation of 9 early cases and 58 control subjects. Using next-generation sequencing and real-time PCR, adeno-associated virus 2 (AAV2), was detected in the plasma of 9/9 and liver of 4/4 patients but in 0/13 sera/plasma of age-matched healthy controls, 0/12 children with adenovirus (HAdV) infection and normal liver function and 0/33 children admitted to hospital with hepatitis of other aetiology. AAV2 typically requires a coinfecting ‘helper’ virus to replicate, usually HAdV or a herpesvirus. HAdV (species C and F) and human herpesvirus 6B (HHV6B) were detected in 6/9 and 3/9 affected cases, including 3/4 and 2/4 liver biopsies, respectively. The class II HLA-DRB1*04:01 allele was identified in 8/9 cases (89%), compared with a background frequency of 15.6% in Scottish blood donors, suggestive of increased susceptibility in affected cases. Acute non-A-E paediatric hepatitis is associated with the presence of AAV2 infection, which could represent a primary pathogen or a useful biomarker of recent HAdV or HHV6B infection. Population and mechanistic studies are required to explore these findings further.