Monitoring Editor: Carole Parent Increased intracellular pH is an evolutionarily conserved signal necessary for directed cell migration. We previously reported that in Dictyostelium cells lacking H(+) efflux by a Na-H exchanger (Ddnhe1(--)), chemotaxis is impaired and the assembly of actin filaments (F-actin) is attenuated. We now describe a modifier screen that reveals the C-terminal fragment of actin interacting protein 1 (Aip1) enhances the chemotaxis defect of Ddnhe1(--) cells but has no effect in wild-type Ax2 cells. However, expression of full-length Aip1 mostly suppresses chemotaxis defects of Ddnhe1(--) cells and restores F-actin assembly. Aip1 functions to promote cofilin-dependent actin remodeling and we found that although full-length Aip1 binds cofilin and F-actin, the C-terminal fragment binds cofilin but not F-actin. Because pH-dependent cofilin activity is attenuated in mammalian cells lacking H(+) efflux by NHE1, our current data suggest that full-length Aip1 facilitates F-actin assembly when cofilin activity is limited. We predict the C-terminus of Aip1 enhances defective chemotaxis of Ddnhe1(--) cells by sequestering the limited amount of active cofilin without promoting F-actin assembly. Our findings indicate a cooperative role of Aip1 and cofilin in pH-dependent cell migration and they suggest defective chemotaxis in Ddnhe1(--) cells is determined primarily by loss of cofilin-dependent actin dynamics.