Extracellular nucleotides are local, short lived signaling molecules that inhibit renal tubular transport via both luminal and basolateral P2 receptors. Apparently, the renal epithelium itself is able to release nucleotides. The mechanism and circumstances under which nucleotide release is stimulated remains elusive. Here, we investigate the phenomenon of nucleotide secretion in intact perfused mouse medullary thick ascending limb (mTAL) and cortical collecting duct (CCD). The nucleotide secretion was monitored by a biosensor adapted to register nucleotides in the tubular out-flow. [Ca2+]i was measured simultaneously in the biosensor cells and the renal tubule with fluo-4. We were able to identified spontaneous tubular nucleotide secretion in resting perfused mTAL. In this preparation 10 nM AVP- and dDAVP induced robust [Ca2+]i oscillations, whereas AVP in the CCD induced large slow and transient [Ca2+]i elevations. Importantly, we identify that AVP/dDAVP triggers tubular secretion of nucleotides in mTAL. After addition of AVP/dDAVP the biosensor register bursts of nucleotides in the tubular perfusate, corresponding to a tubular nucleotide concentration of ~0.2-0.3 mM. A very similar response was observed after AVP stimulation of CCDs. Thus, AVP stimulated tubular secretion of nucleotides in a burst like pattern with peak tubular nucleotide concentrations in the low micro molar range. We speculate that local nucleotide signaling is an intrinsic feed-back element of hormonal control of renal tubular transport.