The outer arm dynein (OAD) complex is the main propulsive force generator for ciliary/flagellar beating. In Chlamydomonas and Tetrahymena, the OAD complex comprises three heavy chains (alpha, beta, and gamma HCs) and > 10 smaller subunits. Dynein light chain-1 (LC1) is an essential component of OAD. It is known to associate with the Chlamydomonas gamma head domain, but its precise localization within the. head and regulatory mechanism of the OAD complex remain unclear. Here Ni-NTA-nanogold labeling electron microscopy localized LC1 to the stalk tip of the gamma head. Single-particle analysis detected an additional structure, most likely corresponding to LC1, near the microtubule-binding domain (MTBD), located at the stalk tip. Pull-down assays confirmed that LC1 bound specifically to the gamma MTBD region. Together with observations that LC1 decreased the affinity of the gamma MTBD for microtubules, we present a new model in which LC1 regulates OAD activity by modulating gamma MTBD's affinity for the doublet microtubule.