The structural organization of different domains of the acrosomal matrix of intact and detergent-extracted rabbit spermatozoa was characterized utilizing both thin sectioning and negative staining techniques. A matrix component with a crystalline substructure was present exclusively within the apical segment of the acrosome. The crystalline matrix remained intact following Triton X-100 extraction and appeared composed of aggregates of plaque-like crystalloids. These crystalloids retained a structural association with a detergent-insoluble coat of the outer acrosomal membrane, termed the acrosomal lamina. Negatively stained specimens demonstrate that the matrix crystalloids are composed of globular subunits, spaced 8-10 nm center-to-center, which are assembled into a two-dimensional lattice. The potential functions of the detergent-insoluble acrosomal lamina-matrix complex are discussed.