Inner dynein arms in cilia and flagella contain actin as a subunit; however, the function of this actin is totally unknown. Here we performed chemical crosslinking experiments to examine the interaction of actin with other subunits. Six of the seven Chlamydomonas inner-arm dynein species separated by anion-exchange chromatography contain actin and, either one of the two previously identified light chains, p28 and centrin, in a mutually exclusive manner. Western blotting of chemically crosslinked dyneins indicated that actin is directly associated with p28 and centrin but not with the dynein heavy chains (HCs). In contrast, p28 and centrin both appeared to interact directly with the N-terminal half of the HCs. Thus it is likely that actin is associated with the heavy chains through p28/centrin. These light chains may well function in the assembly or targeting of the inner arm to the correct axonemal location.