The Dictyostelium discoideum genome has been found to encode three PP2A regulatory subunits (B, B', and B"). In order to study their function, the genes were individually disrupted and the resulting null strains' phenotypes were analyzed. Our results suggested that the PP2A-B' regulatory subunit was essential for proper multicellular development. Additionally, when exposed to a gradient of cAMP, both the PP2A-B and -B' regulatory subunit null strains showed some significant chemotaxis defects. Null strains expressing GFP and the previously knocked out subunits showed that the PP2A-B and -B' subunits are globally distributed throughout chemotaxing cells. Finally, analysis of cAMP-induced F-actin polymerization and myosin assembly revealed that there were noticeable deficits in all of the analyzed null strains' actin profiles (especially that of PP2A-B and -B') when compared to the wild type. Also, there were significant increases in myosin filament formation in PP2A-B". The results suggest that the PP2A-B and -B' regulatory subunits positively regulate F-actin polymerization, although probably through different effector proteins. On the other hand, PP2A-B" appears to negatively regulate myosin filament formation. Therefore, the specificity of PP2A in de-phosphorylating its effector proteins is different depending on the regulatory subunit within the holoenzyme. Also, these differing specificities appear to cause the phosphatase to have unique protein-protein interactions with other effector proteins involved in F-actin polymerization.