5-Azacytidine inhibits DNA synthesis and to a lesser proportion RNA synthesis in S. antibioticus. The biosynthesis of proteins is not affected. The main inhibitory effect of 5-azacytidine on DNA and RNA synthesis is probably caused by its incorporation into newly synthesized DNA or RNA and the formation of covalent complexes between cytosine-specific methyltransferases and the modified DNA or RNA templates. To analyze whether such effects could occur at the oriC region of S. antibioticus we analyzed the methylation status of this region using the bisulphite assisted genomic sequencing method. One of the cytosine residues found to be partially methylated was contained within an unique NaeI sequence (GCCGGC) in oriC. Subsequent analysis shows chromosomal DNA from S. antibioticus to be resistant to R.NaeI restriction indicating that this strain contains a NaeI-specific cytosine C5-methyltransferase activity. Following 5-azacytidine treatment the NaeI site within the oriC region becomes partially demethylated. Our results suggest that some of the 5-azacytidine effects on DNA and RNA synthesis might indeed be related to the complex formation and inhibition of a cytosine-specific DNA methyltransferase.