To examine the effect of transforming growth factor (TGF) beta3 on immortalized myometrial and leiomyoma cell lines cloned from primary cell cultures of surgical specimens, and to determine whether such treatment alters myometrial cell extracellular matrix (ECM) expression.Laboratory study.University hospital.Immortalized myometrial and leiomyoma cells from patients with symptomatic leiomyomata.Tissue culture, followed by cellular, RNA, and protein analysis.Cell proliferation, alteration in ECM component expression.Immortalized leiomyoma and myometrial cells demonstrate increased mRNA and protein production of the ECM proteins, collagen 1A1 (15.0-fold), fibronectin 1 (2.93 fold), and connective tissue growth factor (9.40-fold) with exogenous TGF-beta3 stimulation. Notably, the expression of collagen 1A1, fibronectin 1, and connective tissue growth factor in myometrial cells increase to similar expression levels as those found in leiomyoma cells. In addition, TGF-beta3 decreased production of genes involved in matrix resorption, including matrix metalloproteinase 2 (0.65-fold) and -11 (0.68-fold).TGF-beta3 induced a molecular phenotype in myometrial cells that was similar to leiomyoma cells, with elevated production of ECM-related genes and decreased production of ECM degradation-related genes.