Proliferation of supporting cells in the inner ear is the early major event occurring during hair cell regeneration after acoustic trauma or aminoglycoside treatment. In the present study, we examined the possible influence of 30 growth factors on the proliferation of pure rat utricular epithelial cells in culture. Utricular epithelial sheets were separated and partially dissociated from early postnatal rats via a combined enzymatic and mechanical method. The cultured utricular epithelial cells expressed exclusively epithelial cell antigens, but not fibroblast, glial, or neuronal antigens. With tritiated thymidine incorporation assays, we found that several fibroblast growth factor (FGF) family members, insulin-like growth factor-1 (IGF-1), IGF-2, transforming growth factor-alpha (TGF-alpha), and epidermal growth factor (EGF), stimulated proliferation of the utricular epithelial cells. In contrast, neurotrophins and other growth factors did not elicit any detectable mitogenic effects. Among all of the growth factors examined, FGF-2 was the most potent mitogen. When FGF-2 was added in combination with IGF-1 or TGF-alpha to the medium, combined effects were seen. These results were confirmed with BrdU immunocytochemistry. Thus, the present culture system provides a rapid and reliable assay system to screen novel growth factors involved in proliferation of mammalian inner ear supporting cells. Furthermore, immunostainings revealed that the cultured utricular epithelial cells expressed FGF and IGF-1 receptors, and utricular hair cells produced FGF-2 in vivo. The addition of neutralizing antibodies against FGF-2 or IGF-1 to the cultures significantly inhibited the utricular epithelial cell proliferation. This work suggests that FGF-2 and IGF-1 may regulate the proliferation step during hair cell development and regeneration.