Background: The alignment of two or more protein sequences provides a powerful guide in the prediction of the protein structure and in identifying key functional residues, however, the utility of any prediction is completely dependent on the accuracy of the alignment. In this paper we describe a suite of reference alignments derived from the comparison of protein three-dimensional structures together with evaluation measures and software that allow automatically generated alignments to be benchmarked. We test the OXBench benchmark suite on alignments generated by the AMPS multiple alignment method, then apply the suite to compare eight different multiple alignment algorithms. The benchmark shows the current state-of-the art for alignment accuracy and provides a baseline against which new alignment algorithms may be judged.