Introduction: Muscarinic receptors have been identified both in the suburothelium and urothelium of the human bladder. It has been proposed that increased release of Acetylcholine (ACh) from the urothelial and suburothelial nerves may act on suburothelial and detrusor muscarinic receptors, resulting in detrusor overactivity (DO). Neuropeptide Y (NPY) is a cholinergic co-transmitter. Botulinum neurotoxin type A (BoNT/A) is known to act by blocking the release of ACh. We compared expression of M1, M2, M3 receptors, NPY and SNAP-25 in patients with DO, controls and following successful treatment with BoNT/A. Methods: Flexible cystoscopy bladder biopsies were obtained from 36 patients with DO at baseline, four and sixteen weeks after successful BoNT/A treatment, together with 9 asymptomatic controls. Specimens were immunostained using specific antibodies to the above mentioned antigens. Immunoreactivity (IR) were quantified with image analysis. Results: Reduced levels of M1 IR were noted in DO patients compared to controls. Following BoNT/A, there were increases and „normalisation‟ of M1 IR with similar changes in the urothelium. Significant similar post-BoNT/A increases were seen in M2 IR. Decreased M3 IR was observed at baseline DO compared to controls, with significant increases only in the urothelium following BoNT/A. SNAP-25 IR showed no changes. NPY IR increased in DO, with a decreasing trend following BoNT/A. Inverse IR correlations were found with frequency and urgency. Conclusions: Reduced levels of suburothelial muscarinic receptors in DO are in accordance with previous RT-PCR findings, showing reduced mRNA levels in overactive bladders. NPY IR is increased and may illustrate upregulated cholinergic transmission with DO, similar to ATP. SNAP-25 IR demonstrates the presence of BoNT/A sensitive neurones within the suburothelium. Post-BoNT/A changes in suburothelial muscarinic receptors and NPY appear compensatory to the reduced release of ACh, supporting a neuroplastic effect of BoNT/A on bladder afferent pathways as part of its mechanism of action.