Oocyte-granulosa cell communication, mediated by paracrine factors, is essential for oocyte development. Kit ligand (KITL) is expressed in granulosa cells as soluble (KITL1) or membrane-associated (KITL2) proteins. However, the relative biopotency of each isoform during oocyte development is unknown. Our initial results showed that Kitl2 was down-regulated in cultured granulosa cells. To determine the effect of the two isoforms of KITL on oocyte growth, Kitl-deficient fibroblasts were transfected with constructs expressing either KITL1 or KITL2, and growing oocytes were isolated from 12-day-old mice and cultured on the transfected fibroblasts for 2 days. At the end of culture, oocyte diameters were measured, the incidence of spontaneous germinal vesicle breakdown (GVBD) was noted, and oocytes were analyzed for KIT receptor expression. Oocyte growth occurred only in the presence of the KITL2-producing fibroblasts, and suppression of KITL2 expression impaired oocyte growth. Up-regulation of KIT expression occurred in the presence of KITL2 but not KITL1. The presence of KITL2 inhibited spontaneous GVBD. Meiosis inhibitors did not attenuate the GVBD that occurred in the absence of KITL2, suggesting that this process reflects oocyte degeneration rather than meiotic progression. These results indicate that KITL2 is the principal KITL isoform required for oocyte growth and survival in vitro.