Objectives To characterise separately the pharmacological profiles of spontaneous contractions from the mucosa and detrusor layers of the bladder wall and to describe the relationship in mucosa between adenosine triphosphate (ATP) release and spontaneous contractions. Materials and Methods Spontaneous contractions were measured (36 °C) from isolated mucosa or detrusor preparations, and intact (mucosa + detrusor) preparations from guinea‐pig bladders. Potential modulators were added to the superfusate. The percentage of smooth muscle was measured in haematoxylin and eosin stained sections. ATP release was measured in superfusate samples from a fixed point above the preparation using a luciferin–luciferase assay. Results The magnitude of spontaneous contractions was in the order intact >mucosa >detrusor. The percentage of smooth muscle was least in mucosa and greatest in detrusor preparations. The pharmacological profiles of spontaneous contractions were different in mucosa and detrusor in response to P2X or P2Y receptor agonists, adenosine and capsaicin. The intact preparations showed responses intermediate to those from mucosa and detrusor preparations. Low extracellular pH generated large changes in detrusor, but not mucosa preparations. The mucosa preparations released ATP in a cyclical manner, followed by variations in spontaneous contractions. ATP release was greater in mucosa compared with detrusor, augmented by carbachol and reversed by the M 2 ‐selective antagonist, methoctramine. Conclusions The different pharmacological profiles of bladder mucosa and detrusor, implies different pathways for contractile activation. Also, the intermediate responses from intact preparations implies functional interaction. The temporal relationship between cyclical variation of ATP release and amplitude of spontaneous contractions is consistent with ATP release controlling spontaneous activity. Carbachol‐mediated ATP release was independent of active contractile force.