Toluene is widely used as a component in industrial solvents and many toluene-containing products are abused via inhalation. While many studies have demonstrated its inhibitory effects on neuronal activity, the effects of toluene on receptor signaling in proliferating and differentiating neural precursor cells are presently unclear. Here, using digital video microscopy and Ca2+ imaging, we investigated the effects of acute exposure to toluene on the function of muscarinic acetylcholine receptors (mAChRs) expressed in neural precursor cells. The neural precursor cells were isolatedfrom embryonic day 13 (E13) rat cortex and expanded in serum-free medium containing basic fibroblast growth factor (bFGF). We found that the acetylcholine (ACh) analog carbachol (CCh) induced a dose-dependent increase in cytosolic Ca2+, which was blocked by the muscarinic receptor antagonist atropine in a reversible manner. Toluene was added to the perfusion medium and concentrations of toluene in the medium were determined by gas chromatographic analysis. Following imaging, the cells were fixed and processed for 5-bromo-2'-deoxyuridine (BrdU, cell proliferation marker) and beta-tubulin (TuJ1, neuronal marker) immunostaining. In the 5 day culture, most cells continued to divide (BrdU+), while afew cells differentiated into young neurons (TuJ1-). The CCh-induced Ca2+ elevations in proliferating (BrdU+TuJ1-) neural precursor cells were significantly reduced by acute exposure to 0.15 mM toluene and completely blocked by 10 mM toluene. Toluene's inhibition of muscarinic receptor-mediated Ca2+ signaling was rapid, reversible and dose-dependent with an IC50 value 0.5 mM. Since muscarinic receptors mediate cell proliferation and differentiation during neural precursor cell development, these results suggest that depression of muscarinic signaling may play a role in toluene's teratogenic effect on the developing nervous system.