Activation-induced cytidine deaminase is required for the DNA cleavage step of Ig class switch recombination (CSR). However, its molecular mechanism is controversial. RNA-editing hypothesis postulates that activation-induced cytidine deaminase deaminates cytosine in an unknown mRNA to generate a new mRNA encoding an endonuclease for CSR and thus predicts that DNA cleavage depends on de novo protein synthesis. On the other hand, DNA deamination hypothesis proposes that DNA cleavage is initiated by cytosine deamination in DNA, followed by uracil removal by uracil DNA glycosylase. By using the chromatin immunoprecipitation assay to detect γ-H2AX focus formation as a marker for DNA cleavage, we found that cycloheximide inhibited DNA cleavage in the Ig heavy-chain locus during CSR. Requirement of protein synthesis in the DNA cleavage step of CSR strengthens the RNA-editing hypothesis.