The Escherichia coli AlkB protein is involved in protecting cells against mutation and cell death induced specifically by SN2-type alkylating agents such as methyl methanesulfonate (MMS). A human cDNA encoding a polypeptide homologous to E.coli AlkB was discovered by searching a database of expressed sequence tags (ESTs) derived from high throughput cDNA sequencing. The full-length human AlkB homolog (hABH) cDNA clone contains a 924 bp open reading frame encoding a 34 kDa protein which is 52% similar and 23% identical to E.coli AlkB. The hABH gene, which maps to chromosome 14q24, was ubiquitously expressed in 16 human tissues examined. When hABH was expressed in E.coli alkB mutant cells partial rescue of the cells from MMS-induced cell death occurred. Under the conditions used expression of hABH in skin fibroblasts was not regulated by treatment with MMS. Our findings show that the AlkB protein is structurally and functionally conserved from bacteria to human, but its regulation may have diverged during evolution.