The E.coli DNA repair enzyme AlkB acts by hydroxylating methyl lesions in DNA, such as 1-methylAdenine (1meA) and 3-methylCytosine (3meC), which leads to spontaneous loss of the hydroxymethyl group and recovery of the correct base. Nine different mammalian AlkB homologues have been identified. These proteins belong to the superfamily of 2-oxoglutarate and Fe2+-dependent dioxygenases which include numerous members with a broad assortment of substrates. The aim of this study was to uncover the biological properties of the mammalian E.coli AlkB homologues ALKBH1 and ALKBH4. In paper I we present the first Alkbh4 knockout mouse. Alkbh4-/- mice display embryonic lethality at an early preimplantation stage, and siRNA or Cre-mediated induced ALKBH4 depletion in cell culture results in apoptosis, cytokinesis failure and reduced cell mobility. We provide evidence that ALKBH4 is required for the reversion of mono-methyl modifications on actin lysine-84 (K84me1). ALKBH4 interacts with methylated lysine-84 in actin, and depletion of ALKBH4 in cell culture based studies lead to an increase in the level of actin-K84me1. Methylated K84 is most abundant in F-actin, and when actin-K84 is methylated, the non-muscular myosin II (NMII) displays less ability to bind actin filaments. Based on these findings, we propose a role for ALKBH4 in the regulation of actomyosin dynamics by demethylating actin-K84me1. In paper II, we investigate the physiological role of ALKBH4 in mice using a tamoxifen-inducible Cre-mediated Alkbh4 knockout mice to circumvent embryonic lethality. Surprisingly, the ALKBH4 depleted mice displayed overall similar phenotype as the control animals used in this study, except for a striking failure in the development of promeiotic spermatocytes from the pachytene stage and on. To investigate the role of ALKBH1, we generated mice lacking ALKBH1 by homologous recombination as described in paper III. Lack of ALKBH1 leads to reduced viability during embryonic development, improper development of tissues originating from the ectodermal lineage, sex-ratio distortions and apoptotic haploid cell types in Alkbh1-/- testes.