Homologous recombination (HR) is an essential process in maintaining genome integrity and variability. In eukaryotes, the Rad52 epistasis group proteins are involved in meiotic recombination and/or HR repair. One member of this group, Rad54, belongs to the SWI2/SNF2 family of DNA-stimulated ATPases. Recent studies indicate that Rad54 has important functions in HR, both as a chromatin remodelling factor and as a mediator of the Rad51 nucleoprotein filament. Despite the importance of Rad54 in HR, no study of Rad54 from plants has yet been performed. Here, we cloned the full-length AtRAD54 cDNA sequence; an open reading frame of 910 amino acids encodes a protein with a predicted molecular mass of 101.9 kDa. Western blotting analysis showed that the AtRad54 protein was indeed expressed as a protein of approximately 110 kDa in Arabidopsis. The predicted protein sequence of AtRAD54 contains seven helicase domains, which are conserved in all other Rad54s. Yeast two-hybrid analysis revealed an interaction between Arabidopsis Rad51 and Rad54. AtRAD54 transcripts were found in all tissues examined, with the highest levels of expression in flower buds. Expression of AtRAD54 was induced by γ-irradiation. A T-DNA insertion mutant of AtRAD54 devoid of full-length AtRAD54 expression was viable and fertile; however, it showed increased sensitivity to γ-irradiation and the cross-linking reagent cisplatin. In addition, the efficiency of somatic HR in the mutant plants was reduced relative to that in wild-type plants. Our findings point to an important role for Rad54 in HR repair in higher plants.