DOCK180 is an atypical guanine nucleotide exchange factor of Rac1 identified originally as one of the two major proteins bound to the SH3 domain of the Crk adaptor protein. DOCK180 induces tyrosine phosphorylation of p130(Cas), and recruits the Crk-p130(Cas) complex to focal adhesions. Recently, we searched for DOCK180-binding proteins with a nano-LC/MS/MS system, and found that ANKRD28, a protein with twenty-six ankyrin domain-repeats, interacts with the SH3 domain of DOCK180. Knockdown of ANKRD28 reduced the migration velocity and altered the distribution of focal adhesion proteins such as Crk, paxillin and p130(Cas). On the other hand, the expression of ANKRD28, p130(Cas), Crk and DOCK180 induced hyper-phosphorylation of p130(Cas), which paralleled the induction of multiple long cellular processes. Depletion of ELMO, another protein bound to the SH3 domain of DOCK180, also retarded cell migration, but its expression together with p130(Cas), Crk and DOCK180 induced extensive lamellipodial protrusion around the entire circumference without 130(Cas) hyperphosphorylation. These data suggest the dual modes of DOCK180-Rac regulation for cell migration.