XH/pi hydrogen bonds have been predicted to make important contributions to protein structure and function. NMR evidence is presented for an OH/pi interaction between a highly conserved threonine and phenylalanine pair found specifically in CAP-Gly domains associated with mictrotubule plus ends. The functional contribution of this nonclassical hydrogen bond in target peptide recognition is demonstrated via subtle point mutagenesis. The OH/pi interaction is part of a TxFxxxxW motif that comprises a conserved "threonine clasp" that defines function in CAP-Gly domains.