Oxytocin plays an important role in the progression, timing and modulation of uterine contraction during labor and is widely used as an uterotonic agent. We investigated the mechanisms regulating oxytocin receptor (OTR) signaling in human primary myometrial smooth muscle cells and the ULTR cell-line. Oxytocin produced concentration-dependent increases in both total [³H]-inositol phosphate accumulation and intracellular Ca²⁺ concentration ([Ca²⁺]_i), however responses were greater and more reproducible in the ULTR cell-line. Assessment of phospholipase C activity in single cells revealed that the OTR desensitizes rapidly (within 5 min) in the presence of oxytocin (100 nM). To characterize OTR desensitization further, cells were stimulated with a maximally-effective concentration of oxytocin (100 nM, 30 sec) followed by a variable wash-out period and a second identical application of oxytocin. This brief exposure to oxytocin caused a marked decrease (>70%) in OTR responsiveness to re-challenge and was fully reversed by increasing the time period between agonist challenges. To assess involvement of G protein-coupled receptor kinases (GRKs) in OTR desensitization, cells were transfected with siRNAs to cause specific 75% knockdown of GRKs 2, 3, 5 or 6. In both primary myometrial and ULTR cells knockdown of GRK6 largely prevented oxytocin-induced OTR desensitization; in contrast, selective depletion of GRKs 2, 3 or 5 was without effect. These data indicate that GRK6 recruitment is a cardinal effector of OTR responsiveness and provide mechanistic insight into the likely in vivo regulation of OTR signaling in uterine smooth muscle.