Oestrogens are known to improve flow mediated dilation and reduce myogenic tone in rodents. The mechanism underlying these effects is poorly understood including the relative roles of the different oestrogen receptors (ERα and β). The aim of this study was to characterise the consequence of the loss of ERβ on oestrogen enhanced flow responses and myogenic tone in small arteries from ERβ knock out mice (βERKO) mice and wild type (WT) littermates. Male and female βERKO mice and their wild type littermates were humanly killed by a rising concentration of CO₂ and cervical dislocation. Mesenteric arteries (~185 μm diameter) were dissected and mounted on a pressure myograph (Living Systems Instrumentation, USA). Flow-mediated dilation (9-104μl/min) was assessed in noradrenaline pre-constricted arteries before and after incubation with 17β-oestradiol (3h; 10nM) or, in different vessels, in the presence of the selective ERα agonist PPT (3h; 10nM). Pressure-induced myogenic tone (20-100mmHg) before and after incubation with 17β-oestradiol was also studied. Data are expressed as means ± SEM. Differences between groups were determined by repeated measure ANOVA Flow-mediated dilation increased after incubation with 17β-oestradiol in WT and βERKO females and WT males [before and after incubation with 17β-oestradiol, WT females (n=11) 5.08 ± 2.80 vs 47.1 ± 15.2, βERKO females (n=12) 15.62 ± vs 41.9 ± 12.4 and WT males (n=13) −3.45 ± 5.25 vs 47.2 ± 12.6 % dilation at maximum flow, all P<0.05]. Increased dilation to flow was also observed after incubation with PPT in WT females (9.27 ± 5.81 vs 47.7 ± 15.7 before and after incubation with PPT, n=7, P0.05). Myogenic tone was similar both between groups and between sexes prior to incubation with 17β-oestradiol (P>0.05, n=8 each group). After incubation with 17β-oestradiol a significant decrease in myogenic tone was observed in both WT and βERKO females (18.9 ± 2.81 vs 10.1 ± 0.89 and 12.3 ± 2.42 vs 6.71 ± 1.45 % myogenic tone at 100mmHg before and after incubation with 17β-oestradiol in WT (n=11) and βERKO (n=13) females, respectively, P<0.05) but not in WT or βERKO males (12.2 ± 3.09 vs 7.92 ± 2.24 and 14.9 ± 2.78 vs 8.98 ± 1.86 % myogenic tone at 100mmHg before and after incubation with 17β-oestradiol in WT (n=11) and βERKO (n=11) males, respectively, P<0.05). The absence of ERβ is associated with gender specific alteration in flow-mediated relaxation in response to 17β-oestradiol. Oestrogens, through the action of ERβ are responsible for oestrogen enhanced flow-mediated dilation in male mice whereas in females ERα may mediate this response. Myogenic tone is unaffected by the loss of ERβ in either male or female mice, however gender specific differences exist in myogenic tone after incubation with 17β-oestradiol.