The filamentous fungus Ascobolus immersus represents an eukaryotic model organism to study genetic phenomena linked to DNA methylation. Following our previous characterization of a gene, masc1 from A. immersus, encoding the 'de novo' C5-DNA-methyltransferase (MTase), we report here the identification of a second MTase gene, masc2. The deduced peptide sequence of Masc2 is similar to previously identified eukaryotic MTases and distinct from Masc1 by having a large N-terminal domain in addition to the ubiquitous C-terminal catalytic domain. Following cloning of the gene, Masc2 was overexpressed and purified. Masc2 shows MTase activity with double stranded DNAs. Structural and biochemical properties of Masc2 suggest that it may function as a 'maintenance' MTase. With this finding, A. immersus represents so far the only eukaryotic organism in which two possibly synergistically operating MTases have been identified.