AbstractThe eyes absent (Eya) genes encode a family of proteins that combine the functions of transcriptional cofactors, signal transducers and enzymes, namely protein tyrosine phosphatases. The latter activity resides in the highly conserved C‐terminal Eya domain (ED). Here, we investigated the substrate specificity of the Arabidopsis thaliana homologue (AtEya) by using low‐molecular‐weight compounds and synthetic phosphotyrosine (pY)‐containing peptides that correspond either to phosphorylation sites in proteins or to peptides that were selected through the screening of a combinatorial peptide library. AtEya displayed modest peptide substrate specificity and was sensitive to charges adjacent to pY. In general, the presence of acidic residues on the N‐terminal side of the phosphorylation site was critical for catalysis, whereas basic amino acids seemed to be preferred with respect to high‐affinity binding. We also detected significant acyl phosphatase activity of AtEya; this suggests that Eya proteins might have further substrates in vivo. In addition, we analysed the phosphatase activity of a number of variants of the mouse Eya1 protein that harbours single point mutations that were associated with branchio–oto–renal syndrome (BOR), branchio–oto syndrome (BO) and ocular defects, respectively, in humans. While BOR mutations led to a significantly reduced phosphatase activity, BO mutants as well as those that are associated with ocular defects only displayed activity that was similar to wild‐type levels.