Endocytosis is a major mechanism through which cellular signaling by G protein-coupled receptors (GPCRs) is terminated. However, recent studies demonstrate that GPCRs are internalized in an active state and continue to signal from within endosomes, resulting in effects on cellular function that are distinct to those arising at the cell surface. Endocytosis inhibitors are commonly used to define the importance of GPCR internalization for physiological and pathophysiological processes. Here, we provide the first detailed examination of the effects of these inhibitors on neurogenic contractions of gastrointestinal smooth muscle, a key preliminary step to evaluate the importance of GPCR endocytosis for gut function. Inhibitors of clathrin-mediated endocytosis (Pitstop2, PS2) or G protein-coupled receptor kinase-2/3-dependent phosphorylation (Takeda compound 101, Cmpd101), significantly reduced GPCR internalization. However, they also attenuated cholinergic contractions through different mechanisms. PS2 abolished contractile responses by colonic muscle to SNC80 and morphine, which strongly and weakly internalize δ-opioid and μ-opioid receptors, respectively. PS2 did not affect the increased myogenic contractile activity following removal of an inhibitory neural influence (tetrodotoxin) but suppressed electrically evoked neurogenic contractions. Ca²⁺ signaling by myenteric neurons in response to exogenous ATP was unaffected by PS2, suggesting inhibitory actions on neurotransmitter release rather than neurotransmission. In contrast, Cmpd101 attenuated contractions to the cholinergic agonist carbachol, indicating direct effects on smooth muscle. We conclude that, although PS2 and Cmpd101 are effective blockers of GPCR endocytosis in enteric neurons, these inhibitors are unsuitable for the study of neurally mediated gut function due to their inhibitory effects on neuromuscular transmission and smooth muscle contractility. NEW & NOTEWORTHY Internalization of activated G protein-coupled receptors is a major determinant of the type and duration of subsequent downstream signaling events. Inhibitors of endocytosis effectively block opioid receptor internalization in enteric neurons. The clathrin-dependent endocytosis inhibitor Pitstop2 blocks effects of opioids on neurogenic contractions of the colon in an internalization-independent manner. These inhibitors also significantly impact cholinergic neuromuscular transmission. We conclude that these tools are unsuitable for examination of the contribution of neuronal G protein-coupled receptor endocytosis to gastrointestinal motility.