FAP52 is a recently described focal adhesion-associated protein. It is a member of an emerging PCH (pombe Cdc15 homology) family of proteins characterized by a common domain organization and involvement in actin cytoskeleton organization, cytokinesis, and vesicular trafficking. Using gel filtration, surface plasmon resonance, and native polyacrylamide gel electrophoresis analysis, combined with chemical cross-linking of both native and recombinant protein, we show that FAP52 self-associates in vitro and suggest that it occurs predominantly as a trimer also in vivo. Analysis of the various domains of FAP52 by surface plasmon resonance showed that the highly alpha-helical region in the N-terminal half of the protein provides the self-association interface. Overexpression of the oligomerization domain in cultured cells was accompanied by major alterations in cellular morphology, actin organization, and the structure of focal adhesions, suggesting that an orderly coming together of FAP52 molecules is crucial for a proper actin filament organization and cytoskeletal structure. Comparison of the primary structures shows that all of the members of the PCH family have, in their N-terminal halves, a similar, highly alpha-helical region, suggesting that they all have a capacity to self-associate.