The effect of Zn(II) binding on the structure of bovine alpha-lactalbumin (LA) was investigated. alpha-Lactalbumin, a regulatory subunit of lactose synthase, binds Ca(II) and Zn(II) at different sites in a mutually non-exclusive manner. The structures of the metal-depleted form of LA (apo-LA) and Ca(II)-bound LA (holo-LA) have been well characterized. Here, the effect of Zn(II) binding on the structure of holo-LA has been investigated by comparison with the structure of holo-LA and apo-LA using CD and NMR spectroscopy. The CD spectrum of Zn(II)-holo-LA was similar to that of holo-LA, but the intensity of the negative peak in near-UV region was decreased. Zn(II) binding to holo-LA produced only small changes in NMR chemical shifts, but the integral volumes of the cross-peaks of NOESY signals in cluster II, which is in the vicinity of Zn(II) binding site, were affected. Zn(II) binding induces a local structural change on the holo-LA, but it does not induce a large backbone conformational change.