Background: Substrate specificity determinants of mammalian haloacid dehalogenase (HAD) phosphatases are poorly understood. Results: AUM (aspartate-based, ubiquitous, Mg 2ϩ -dependent phosphatase) is a novel tyrosine phosphatase and paralog of the serine/threonine-and pyridoxal 5Ј-phosphate phosphatase chronophin. Conclusion: Conserved cap residues in AUM or chronophin determine phosphatase substrate specificity. Significance: These findings provide a starting point for structure-based development of HAD phosphatase inhibitors.