The dynein motor domain consists of a ring of six AAA domains with a protruding microtubule-binding stalk and a C-terminal domain of unknown function. To understand how conformational information is communicated within this complex structure, we produced a series of recombinant and proteolytic rat motor domain fragments, which we analyzed enzymatically. A recombinant 210-kDa half-motor domain fragment surprisingly exhibited a 6-fold higher steady state ATPase activity than a 380-kDa complete motor domain fragment. The increased ATPase activity was associated with a complete loss of sensitivity to inhibition by vanadate and an approximately 100-fold increase in the rate of ADP release. The time course of product release was discovered to be biphasic, and each phase was stimulated approximately 1000-fold by microtubule binding to the 380-kDa motor domain. Both the half-motor and full motor domain fragments were remarkably resistant to tryptic proteolysis, exhibiting either two or three major cleavage sites. Cleavage near the C terminus of the 380-kDa motor domain released a 32-kDa fragment and abolished sensitivity to vanadate. Cleavage at this site was insensitive to ATP or 5'-adenylyl-beta,gamma-imidodiphosphate but was blocked by ADP-AlF3 or ADP-vanadate. Based on these data, we proposed a model for long range allosteric control of product release at AAA1 and AAA3 through the microtubule-binding stalk and the C-terminal domain, the latter of which may interact with AAA1 to close the motor domain ring in a cross-bridge cycle-dependent manner.