The AlkA protein from the archaebacterium Archaeglobus fulgidus was characterised with respect to release of hypoxanthine from DNA. The hypoxanthine glycosylase activity had optimal activity at 60 degrees C at pH 5.0. The enzyme released hypoxanthine from substrates with a preference for dI:dG >> dI:dT > dI:dC > dI:dA. The presence of a mismatch on either side of the dIMP in the substrate reduced excision efficiency of the hypoxanthine residue at neutral pH, while a mismatch on both sides of the dIMP resulted in total loss of excision. Release of hypoxanthine from DNA required a minimum of two bases on the 5' side and four bases on the 3' side of the dIMP residue.