Leiomyomas exhibit a large degree of extracellular matrix (ECM) disorganization. Versican, a hyaluronan-chondroitin sulfate binding glycosaminoglycan (GAG), forms an integral part of the ECM and serves as a repository for growth factors important for leiomyoma proliferation. Versican has four splice variants which express different amounts of GAGs. Versican variant, V0, contains the largest amount of GAGs. Using myometrial and leiomyoma tissue and immortalized cell cultures, we hypothesized that V0 expression would be regulated by TGF-β3, which has been implicated in numerous fibrotic diseases. Laboratory study. After IRB approval, we obtained spontaneous uterine leiomyoma and matched myometrium from hysterectomy specimens. Primary cell cultures were generated and immortalized using recombinant retrovirus containing the E6/E7 open reading frames of HPV type-16. Cells were grown to 80% confluence, serum-starved for 48 hours, and then treated for 24 hours with various concentrations of TGF-β3 and Anti-TGF-β3. V0 expression was analyzed by end point RT-PCR and quantitative RT-PCR in both tissue and immortalized cell cultures. V0 was upregulated in leiomyoma vs. myometrial tissue (mean 3.61, Wilcoxon signed rank P<0.05) and cell culture (mean 2.96, P<0.05). With the addition of TGF-β3, myometrial cells demonstrated increased relative fold expression of V0 compared to untreated myometrial expression (1.65, 34.5, and 11.4, P<0.05 at 0.1 ng/ml, 1.0 ng/ml, and 10 ng/ml of TGF-β3). Leiomyoma cells exhibited a less pronounced stimulatory effect of TGF-β3 compared to untreated myometrial cells (2.48, 12.9, and 2.62, P<0.05 at 0.1 ng/ml, 1.0 ng/ml, and 10 ng/ml of TGF-β3). The addition of Anti-TGF-β3 decreased the relative fold expression of leiomyomas cells compared to untreated leiomyomas cells (0.27, 0.20, and 0.02 at 0.1 ug/ml, 1.0 ug/ml, and 10 ug/ml of Anti-TGF-β3). The GAG-rich V0 is overexpressed in leiomyoma vs. myometrial tissue and immortalized cell culture. The addition of TGF-β3 stimulated the expression of V0 in both myometrial and leiomyoma cells, while Anti-TGF-β3 decreased the expression of V0 in leiomyomas cells. Differing levels of GAG composition and amount in leiomyomas contribute to the disorganized ECM by obstructing the tight packing of collagen fibrils. Our data suggest that the larger GAG-rich V0 affects ECM composition and is important to the leiomyoma phenotype.