AbstractAnalysis of Chlamydomonas axonemes revealed that the protein phosphatase, PP2A, is localized to the outer doublet microtubules and is implicated in regulation of dynein‐driven motility. We tested the hypothesis that PP2A is localized to the axoneme by a specialized, highly conserved 55‐kDa B‐type subunit identified in the Chlamydomonas flagellar proteome. The B‐subunit gene is defective in the motility mutant pf4. Consistent with our hypothesis, both the B‐ and C‐ subunits of PP2A fail to assemble in pf4 axonemes, while the dyneins and other axonemal structures are fully assembled in pf4 axonemes. Two pf4 intragenic revertants were recovered that restore PP2A to the axonemes and re‐establish nearly wild‐type motility. The revertants confirmed that the slow‐swimming Pf4 phenotype is a result of the defective PP2A B‐subunit. These results demonstrate that the axonemal B‐subunit is, in part, an anchor protein required for PP2A localization and that PP2A is required for normal ciliary motility. © 2011 Wiley‐Liss, Inc.