The human uterus contains large numbers of mast cells located in close proximity to the myometrial smooth muscle cells (Sivridis et al., 2001), suggesting that they may be a target tissue. Mast cells release a wide variety of inflammatory mediators such as histamine, which causes weak uterine contractions (Cruz et al., 1989). We have previously demonstrated that histamine stimulated Ca²⁺ signalling in ULTR cells, an immortalized human uterine smooth muscle cell line, occurs via the H₁ receptor (Willets et al., 2007). In this study we have used confocal imaging of the fluorescent biosensor eGFP-PH_PLCδ (Willets et al., 2005) to assess the regulation of H₁ histamine receptor phospholipase C (PLC)-stimulated signalling in ULTR cells. Histamine (1μM to 1mM) produced rapid but transient, concentration-dependent translocations of eGFP-PH_PLCδ from the plasma membrane to the cytoplasm, indicative of IP₃ production. To quantify H₁ receptor desensitization ULTR cells were stimulated with an approximate EC₅₀ concentration of histamine (10 μM, for 30 sec) before (R1) and after (R2) exposure to maximal agonist concentration (Rmax, 100 μM) for 60 sec, with 5 min washes between challenges. This protocol produced a significant attenuation of the R2 response compared to R1 (34 ± 6%, n = 7; means ± SEM, p<0.01; one-way ANOVA/Bonferroni’s multiple comparison test), indicative of receptor desensitization. R2 responses returned to the R1 level when cells were washed for 15 min after maximal agonist challenge. To determine whether G protein-coupled receptor kinases were involved in H₁ receptor desensitization, ULTR cells were transfected with siRNA (10 nM) against GRK2 (5’-GGCAGCCAGUGACCAAAAAtt- 3’), GRK6 (5’-GGACACAAAAGGAAUCAAGtt-3’), or a negative control siRNA (Ambion, UK) for 48 h. Endogenous GRK2 and GRK6 expression was reduced by (80 ± 2 %, n = 4 and 83 ± 5 % n = 4, respectively), while the negative control siRNA had no effect. Using the R1/Rmax/R2 protocol, siRNA-mediated suppression of GRK2 almost completely prevented H₁ receptor desensitization, whilst GRK6 depletion was ineffective. Further studies showed that siRNA-mediated GRK2 depletion did not affect the desensitization of endogenously expressed B₂ bradykinin and type 1 angiotensin II receptors. These data suggest that endogenous GRK2 may play an important role in the regulation of H₁ histamine receptor signalling in human uterine smooth muscle.