Endocannabinoids are small signaling lipids, with 2-arachidonoylglycerol (2-AG) implicated in modulating axonal growth and synaptic plasticity. The concept of short-range extracellular signaling by endocannabinoids is supported by the lack of trans-synaptic 2-AG signaling in mice lacking sn-1-diacylglycerol lipases (DAGLs), synthesizing 2-AG. Nevertheless, how far endocannabinoids can spread extracellularly to evoke physiological responses at CB₁ cannabinoid receptors (CB₁Rs) remains poorly understood. Here, we first show that cholinergic innervation of CA1 pyramidal cells of the hippocampus is sensitive to the genetic disruption of 2-AG signaling in DAGLα null mice. Next, we exploit a hybrid COS-7-cholinergic neuron co-culture system to demonstrate that heterologous DAGLα overexpression spherically excludes cholinergic growth cones from 2-AG-rich extracellular environments, and minimizes cell-cell contact in vitro. CB₁R-mediated exclusion responses lasted 3 days, indicating sustained spherical 2-AG availability. Overall, these data suggest that extracellular 2-AG concentrations can be sufficient to activate CB₁Rs along discrete spherical boundaries to modulate neuronal responsiveness.