DNA damage and repair in trypanosomatids impacts virulence, drug resistance and antigenic variation but, currently, little is known about DNA damage responses or cell cycle checkpoints in these divergent protozoa. One of the earliest markers of DNA damage in eukaryotes is ?H2A(X), a serine phosphorylated histone H2A (variant). Here, we report the identification and initial characterization of ?H2A in Trypanosoma brucei . We identified Thr 130 within the replication-dependent histone H2A as a candidate phosphorylation site and found that the abundance of this trypanosomal ?H2A increased in vivo in response to DNA damage. Nuclear ?H2A foci mark the sites of putative natural replication fork stalling, sites of meganuclease-induced DNA double strand breaks and sites of methyl methanesulphonate-induced DNA damage. Naturally occurring and meganuclease-induced ?H2A and RAD51 double-positive repair foci are typically found in S-phase or G 2 nuclei. The results link trypanosomal ?H2A, with an unusual histone modification motif, to DNA damage sensing and mitotic checkpoint signaling. (C) 2012 Elsevier B.V. All rights reserved.