The tyrosine kinase pp60(c-src) has been implicated as a regulator of focal adhesion formation and cell spreading. Here we show that c-src also regulates cell motility and is a key component in the signaling pathway triggered by the motogenic hyaluronan receptor RHAMM, which has been shown to regulate focal adhesion turnover and to regulate ras. Fibroblasts derived from mice lacking src, (src (-/-)), have a random locomotion rate that is significantly slower than the corresponding wild-type fibroblasts. Cell locomotion in these mutant cells is restored by the expression of c-src containing a functional kinase domain, but not by the expression of a kinase-deficient src or by a truncated src containing only functional SH2 and SH3 domains. RHAMM is also required for the restoration of src (-/-) cell locomotion. Thus, the motility of cells expressing c-src is reduced to src (-/-) levels by anti-RHAMM blocking antibodies while the cell locomotion of src (-/-) fibroblasts remains unaffected by anti-RHAMM antibodies. We predict that src acts downstream of RHAMM in the regulation of motility, since the expression of a dominant negative src significantly inhibits RHAMM-dependent ras and serum regulated cell locomotion, the expression of v-src enhances cell motility in a RHAMM independent fashion, and there is a physical and functional assocation between src and RHAMM in ras-transformed cells. However, we suggest that RHAMM regulates focal adhesion turnover via additional src-independent mechanisms. Thus, v-src is unable to turnover focal adhesions in the absence of RHAMM. These results directly demonstrate for the first time a role for src in the regulation of cell locomotion and confirm a key and complex role for src in the regulation of the actin cycle.