The kinesin-1 motor protein transports intracellular cargo to the plus ends of microtubules. In cells, the majority of kinesin-1 exists in a regulated state that binds ADP tightly and has weak microtubule affinity. Regulation involves a direct interaction between the enzymatically active head domains and the regulatory tail domains. This interaction has been shown to inhibit both intrinsic and microtubule-stimulated ADP release, although the mechanism of inhibition is unknown. Here, we use electron paramagnetic resonance and fluorescence spectroscopy to study the kinesin-1 head-tail interaction. We show that the probe mobility of two different spin-labeled nucleotide analogs (2′3′-SLADP and SSL-NANDP) in the kinesin-1 nucleotide pocket is restricted upon addition of exogenous tail domains to truncated kinesin-1 heads. This tail-induced conformational restriction is distinct from the “closing” of Switch I that is observed when kinesin-1 binds microtubules. Unlike myosin V, the head-tail interaction is not nucleotide-dependent, and our data demonstrate that the interaction can occur in the absence of an intact gamma-phosphate sensor. Additionally, we find that the head-tail interaction does not stabilize Mg2+ in the nucleotide pocket. The conformational restriction also occurs when a tail construct containing a K922A point mutation is used. This mutation eliminates the tail's ability to inhibit ADP release, indicating that the tail does not inhibit nucleotide ejection from the pocket by simple steric hindrance. Our combined data support a mechanism in which the tail forms interactions around the nucleotide pocket and acts as a structural support, positioning the critical K922 residue to exert its inhibitory effect. By ruling out various other mechanisms, we propose that K922 may inhibit ADP release by interacting with the nucleotide alpha/beta-phosphates in a manner analogous to the arginine finger regulators of some G-proteins.