Abstract Prokineticins are novel peptides with reported effects on gastrointestinal contractility. Prokineticin actions are mediated by distinct prokineticin receptors (PKR1 and PKR2). This study investigated the role of prokineticin 1 in colonic contractility as well as sites of expression of its receptor in the mouse proximal colon by immunohistochemistry and confocal microscopy. Prokineticin 1 suppressed giant contractions in circular muscle. The inhibitory effect of prokineticin 1 on giant contractions was blocked by the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME). In vitro, prokineticin 1 stimulated nitric oxide release from longitudinal muscle-myenteric plexus cultures. This effect was blocked by l-NAME. PKR1 is expressed on myenteric plexus neurons and colocalizes with a small subset of nNOS expressing neurons. This study suggests that PKR1 mediates an inhibitory effect in vitro, most likely through direct or indirect stimulation of nitric oxide release. PKR1 and its natural ligand, prokineticin 1 may be important for modulation of colonic motility.