Uterine leiomyomas are benign myometrial neoplasms that function as one of the common indications for hysterectomy. Clinical and biological evidences indicate that uterine leiomyomas are estrogen-dependent. Estrogen stimulates cell proliferation through binding to the estrogen receptor (ER), of which both subtypes alpha and beta are present in leiomyomas. Clinically, leiomyomas may be singular or multiple, where the first one is rarely recurring if removed and the latter associated to a relatively young age or genetic predisposition. These markedly different clinical phenotypes indicate that there may different mechanism causing a similar smooth muscle response. To investigate the relative expression of ER alpha and ER beta in multiple and solitary uterine leiomyomas, we collected samples from 35 Chinese women (multiple leiomyomas n = 20, solitary leiomyoma n = 15) undergoing surgery to remove uterine leiomyomas. EUSA assay was performed to detect estrogen(E-2) concentration. Quantitative real-time PCR analysis was performed to detect ER alpha and ER beta mRNA expression. Western blot and immunohistochemical analysis were performed to detect ER alpha and ER beta protein expression. We found that ER alpha mRNA and protein levels of in multiple leiomyomas were significantly lower than those of solitary leiomyomas, whereas ER beta mRNA and protein levels in multiple leiomyomas were significantly higher than those in solitary leiomyomas, irrespectively of the menstrual cycle stage. In both multiple and solitary leiomyomas, ER alpha expression was higher than that of ER beta. E-2 concentration in multiple and solitary leiomyomas correlated with that of ER alpha expression. ER alpha was present in nuclus and cytoplasma while estrogen receptor beta localized only in nuclei in both multiple and solitary leiomyomas. Our findings suggest that the difference of ER alpha and ER beta expression between multiple and solitary leiomyomas may be responsible for the course of the disease subtypes. (C) 2015 Elsevier Inc. All rights reserved.