The inhibition of the enzyme dipeptidyl-peptidase IV (DPP-IV) is an effective pharmacotherapeutic approach for the management of type 2 diabetes. Recent findings have suggested that dietary proteins, including bovine alpha-lactalbumin, could be precursors of peptides able to inhibit DPP-IV. However, information on the location of active peptide sequences within the proteins is far from being comprehensive. Moreover, the traditional approach to identify bioactive peptides from foods can be tedious and long. Therefore, the objective of this study was to use peptide arrays to screen alpha-lactalbumin-derived peptides for their interaction with DPP-IV. Deca-peptides spanning the entire alpha-lactalbumin sequence, with a frame shift of 1 amino acid between successive sequences, were synthesized on cellulose membranes using "SPOT" technology, and their binding to and inhibition of DPP-IV was studied. Among the 114 alpha-lactalbumin-derived decamers investigated, the peptides (60)WCKDDQNPHS(69) (alpha K-i = 76 mu M), (105)LAHKALCSEK(114) (K-i = 217 mu M) and (110)LCSEKLDQWL(119) (K-i = 217 mu M) were among the strongest DPP-IV inhibitors. While the SPOT-and traditionally-synthesized peptides showed consistent trends in DPP-IV inhibitory activity, the cellulose-bound peptides' binding behavior was not correlated to their ability to inhibit the enzyme. This research showed, for the first time, that peptide arrays are useful screening tools to identify DPP-IV inhibitory peptides from dietary proteins.