Human papilloma virus (HPV) infection is considered as an important aetiological factor for anal squamous cell carcinoma (ASCC) but is not sufficient for tumour progression. This carcinoma is poorly understood at the molecular level. Using the largest cohort of cases to date we investigated the molecular mechanisms underlying ASCC development, in particular the roles of TP53, MDM2 and AKT. Viral infection in our cohort occured at high frequency (73%, 94/128) with HPV16 accounting for the majority (86%, 81/94) of infected cases. Only 4% (5/119) of ASCCs showed TP53 (exons 5–8) mutations, but a high frequency (91%, 100/110) of nuclear protein expression of TP53 was observed. There was a significant association (p < 0.001) between nuclear accumulation of TP53 and MDM2 protein although no MDM2 mutations were found, and copy number was normal. Cellular accumulation of phosphorylated-AKT was observed in 66% (82/125) of ASCCs and an association demonstrated between nuclear accumulation of MDM2 and activated AKT (p < 0.001). We observed a high frequency of copy number gain at PIK3CA (47%), and some coding sequence mutations (4%). Amplification of PIK3CA was associated with presence of phosphorylated-AKT (p= 0.008). There was no association between virus infection and TP53 nuclear accumulation (p = 0.5). However, a significant association was found between infection and MDM2 nuclear staining, and between infection and activated AKT (p = 0.04, p = 0.01, respectively). We propose that activation of AKT, possibly through the PI3K-AKT pathway, is an important component of ASCC tumorigenesis that contributes to MDM2 and TP53 accumulation in the nucleus. © 2007 Wiley-Liss, Inc.