Intermolecular interactions between the cytoskeletal proteins talin and vinculin are required for outside-in integrin signaling triggered by the formation of focal adhesions. Talin possesses three non-contiguous vinculin-binding sites (VBS); binding of one of these motifs, VBS3, provokes dramatic alterations in the structure of the vinculin's head (Vh) domain and this activates vinculin (Izard et al., 2004). To address the role of talin's other VBSs in vinculin activation, talin VBS1 (human residues 607-636) was crystallized in complex with the Vh (human residues 1-258) domain. Rhombohedral crystals of human Vh-VBS1 were obtained. The crystals belong to space group R32, with unit-cell parameters a = 88.7 A, alpha = 105.5 degrees and diffract to 2.4 A on a third-generation synchrotron source. The packing density for one heterodimer in the asymmetric unit is 3.04 A(3) Da(-1), with a solvent content of 0.59.