Extracellular matrix plays a key role in cell growth, survival, and differentiation in a wide array of tissue types through integrin-mediated signaling pathways and its interaction with growth factors. This study investigates the role of extracellular matrix and its interaction with activin-A on in vitro growth and survival of mouse preantral follicles. Preantral follicles isolated from 14-day-old immature mouse ovaries were cultured either 3 dimensionally using basement membrane matrix (growth factor - reduced matrigel) or 2 dimensionally on cover slips coated with a single component of extracellular matrix (fibronectin, collagen, or laminin), on polylysine (negative control), or in standard culture plates in a serum-free culture medium with or without activin-A for 7 days. Follicles cultured in matrigel maintained well their 3-dimensional structure compared to those cultured conventionally. This observation was confirmed by analyzing 3-dimensional images of follicles cultured in matrigel and standard culture plate using confocal microscopy. Furthermore, follicles displayed higher growth and survival rates and exhibited antral space formation as early as day 5 of culture when activin-A was added to matrigel; in contrast, the addition of activin-A had no effect on the growth and survival of follicles cultured on individual extracellular matrix components after 7 days of culture. These data may suggest that 3-dimensional culture with extracellular matrix and activin-A provides a better milieu for in vitro growth and survival of preantral follicles in immature mice.