Peristaltic waves of the ureteric smooth muscles move urine down from the kidney, a process that is commonly defective in congenital diseases. To study the mechanisms that control the initiation and direction of contractions, we used video microscopy and optical mapping techniques and found that electrical and contractile waves began in a region where the renal pelvis joined the connective tissue core of the kidney. Separation of this pelvis–kidney junction from more distal urinary tract segments prevented downstream peristalsis, indicating that it housed the trigger for peristalsis. Moreover, cells in the pelvis–kidney junction were found to express isoform 3 of the hyperpolarization-activated cation on channel family known to be required for initiating electrical activity in the brain and heart. Immunocytochemical and real-time PCR analyses found that hyperpolarization-activated cation-3 is expressed at the pelvis–kidney junction where electrical excitation and contractile waves originate. Inhibition of this channel caused a loss of electrical activity at the pelvis–kidney junction and randomized the origin of electrical activity in the urinary tract, thus markedly perturbing contractions. Collectively, our study demonstrates that hyperpolarization-activated cation-3 channels play a fundamental role in coordinating proximal-to-distal peristalsis of the upper urinary tract. This provides insight into the genetic causes of common inherited urinary tract disorders such as reflux and obstruction.