RHO GTPases are small monomeric proteins that play a key role in the regulation of the actin cytoskeleton. In human myometrium, the RHOA–RHO kinase pathway in response to oxytocin and other stimulatory agonists can inhibit myosin light chain phosphatase to produce a calcium-independent increase in myosin light chain phosphorylation and tension [1,2]. Transfection of activated RHOA and RHOB GTPases into quiescent fibroblasts induces the formation of actin stress fibres and adhesion complexes [3,4]. Thus RHOB is a potential regulator of smooth muscle contraction. We aim to quantify the presence and localisation of RHOA and RHOB GTPases in non-pregnant (NP), pregnant at term not in labour (NIL), pregnant at term in labour (SL) and spontaneous preterm labouring human myometrium using immunoblotting and immunohistochemistry. Four individual samples from each group will be analysed. Expression of their related effector proteins (DIAPH1, DIAPH2, ROCK1 and ROCK2) will be determined using immunoblotting and densitometry. mRNA will be extracted from myometrial tissue, differential expression of RHOA and RHOB mRNA will be determined using SYBR green detection and normalised to RPII RNA polymerase II (house keeping gene) expression in NP tissue. RHOA and RHOB GTPase protein expression was similar in the NP, NIL, SL and SPT samples. DIAPH1, DIAPH2, ROCK1 and ROCK2 were also expressed in myometrial tissues with no change in expression between the four groups of samples examined. There was a two-fold increase in RHOA and RHOB mRNA expression in pregnant relative to non-pregnant myometrium; however, this was not significant by analysis by ANOVA (RHOA 95% CI -0.2029 to 2.906 (p>0.05); RHOB 95% CI -0.4570 to 2.652 (p>0.05). Immunohistochemical analysis from the four individual samples revealed that RHOB GTPase has a cytoplasmic distribution in myometrial cells, with staining localised to the perinuclear region in endometrial cells. To our knowledge, this is the first time RHOB GTPase mRNA and protein expression has been described in human myometrium. This is also the first characterisation of known effectors of RHOB (mDia1 and mDia 2) in human myometrial tissue samples. RHOA and RHOB GTPase protein expression is similar in non-pregnant, pregnant preterm and term labouring myometrium. We aim to further explore RHOB GTPase function in myometrial contraction using agonist stimulation and pull down assays.