In this study we performed a systematic comparative analysis of two culture environments-flat/adhesive liquid and three-dimensional collagen gel-upon in vitro ovarian follicle development. We paid particular attention to the effects of in vitro environments upon the preservation of follicular structure and of peri- and intra-follicular extracellular matrix. We show that flat/adhesive environment leads to an obvious distortion of follicle morphology, marked extracellular matrix modifications and high rates of spontaneous, i.e., FSH-independent, follicle disruption. In contrast, three-dimensional collagen gel environments are able to maintain follicular structure with an in vivo-like basal lamina architecture, minimizing spontaneous disruption. Follicle distortions found in flat/adhesive culture systems include a pronounced flattening, causing the follicle horizontal diameters not to adequately reflect follicle volume. Our volume data, based on three-axis follicle diameter measurements, indicate that three-dimensional collagen gel environments increase follicle growth, particularly in response to FSH. This study demonstrates that preservation of both peri- and intra-follicular extracellular matrix compartments during the in vitro growth and differentiation of ovarian follicles is highly desirable, and is now possible through the use of appropriate three-dimensional collagen gel culture environments. This system allows a better understanding of the specific roles played by each of the follicle compartments during development.