It is thought that the growth of uterine leiomyomas may be mediated by the interaction of estrogen receptor alpha (ERalpha) and growth factor pathways and that phosphorylation of ERalpha at serine 118 (ERalpha-phospho-Ser118) is important in this interaction. In this study, immunoblotting and immunohistochemistry were used to investigate the expression of ERalpha-phospho-Ser118, phosphorylated p44/42 mitogen-activated protein kinase (phospho-p44/42 MAPK), and proliferating cell nuclear antigen (PCNA) in human leiomyoma and myometrial tissues during the proliferative and secretory phases of the menstrual cycle. We found that tumors taken from the proliferative phase expressed significantly higher levels of ERalpha-phospho-Ser118, phospho-p44/42 MAPK, and PCNA compared to patient-matched myometria and had significantly higher ERalpha-phospho-Ser118 and PCNA expression compared to secretory phase tumors. Also, enhanced colocalization and association of phospho-p44/42 MAPK and ERalpha-phospho-Ser118 were observed in proliferative phase tumors by confocal microscopy and immunoprecipitation, respectively. These data suggest that ERalpha-phospho-Ser118 may be important in leiomyoma growth and is possibly phosphorylated by phospho-p44/42 MAPK.