ATP release from urinary bladder is vital for afferent signaling. The aims of this study were to localize CALHM1 and pannexin-1 expression and to determine their involvement in mediating ATP release in the bladder. Gene expression by PCR and immunohistochemistry were performed in the porcine bladder. CALHM1 and pannexin-1 mediated ATP release in response to hypotonic solution (0.45% NaCl) induced stretch and extracellular Ca²⁺ depletion ([Ca²⁺]0) was measured in isolated urothelial, suburothelial and detrusor muscle cells. CALHM1 and pannexin-1 mRNA and immunoreactivity were detected in urothelial, suburothelial and detrusor muscle layers with a highest expression on urothelium. Hypotonic stretch caused a 2.7-fold rise in ATP release from all three cell populations (P < 0.01), which was significantly attenuated by the pannexin-1 inhibitor, ¹⁰Panx1, and by CALHM1 antibody. Brefeldin A, a vesicular transport inhibitor, and ruthenium red, a non-selective CALHM1 channel blocker, also significantly inhibited stretch-mediated ATP release from urothelial cells. [Ca²⁺]0 caused a marked but transient elevation of extracellular ATP level in all three cell populations. CALHM1 antibody and ruthenium red inhibited [Ca²⁺]0-induced ATP release from urothelial cells, but their effects on suburothelial and detrusor cells was insignificant. ¹⁰Panx1 showed no significant inhibition of [Ca²⁺]0-induced ATP release in any types of cells. The results presented here provide compelling evidence that pannexin-1 and CALHM1 that are densely expressed in the porcine bladder function as ATP release channels in response to bladder distension. Modulation of extracellular Ca²⁺ may also regulate ATP release in the porcine bladder through voltage gated CALHM1 ion channels.