The Ral family of small G proteins has been implicated in tumorigenesis, invasion, and metastasis. However, little emphasis has been placed on clarifying the individual roles of the two Ral proteins, RalA and RalB, in these processes in view of their high sequence homology. Here we analyze the separate contributions of RalA and RalB in regulating cell migration, a necessary component of the invasive phenotype, in two human cancer cell lines; UMUC-3, a bladder carcinoma line, and the prostate carcinoma line, DU145. Although inhibiting RalA protein expression by approximately 80% with two different small interfering RNA duplexes had no effect on migration, inhibiting RalB expression to the same extent with two different duplexes resulted in a marked reduction in migration. Inhibiting RalB expression did trigger a significant loss of actin cytoskeleton fibers in UMUC-3 that was not seen with inhibition of RalA expression. Interestingly, simultaneous inhibition of RalA and RalB expression had no effect on migration. However, dual inhibition of RalA and RalB expression in UMUC-3 did result in an almost total loss of actin fibers as well as a reduction in proliferation, particularly in reduced serum conditions. These results suggest that RalA and RalB have different roles in cell migration and that they may in fact act as antagonists with regard to this phenotype. As further verification of this hypothesis, we found that expression of constitutively active RalA inhibited migration, whereas expression of constitutively active RalB stimulated migration, consistent with this model. In summary, we present the first demonstration that despite their significant sequence homology, RalA and RalB have nonoverlapping and opposing functions in cancer cell migration but overlapping functions in cell growth.