Summary. Acrosin and its zymogen form proacrosin were located in various sperm fractions by biochemical and immunocytochemical techniques, using an anti-acrosin serum that cross-reacts strongly with proacrosin. If activation of proacrosin was prevented, the zymogen was associated almost entirely with the sperm heads, where it was confined to the anterior segment of the acrosome. Electron microscopy revealed that the acrosomal lumen of such heads remained full of matrix material, and the outer acrosomal membrane remained closely apposed to the other head structures. However, if activation had been allowed to take place, the resultant acrosin and the remaining proacrosin were associated with all sperm fractions and no specific location was observed. In these circumstances, the matrix material was almost entirely absent from the heads, and the outer acrosomal membrane, though usually still present, was only loosely attached. It is concluded that (1) neither acrosin nor proacrosin are truly membrane-bound; they behave as 'diffusible' or partly soluble proteins, although they display a non-specific affinity for cell membrane and other surfaces; (2) proacrosin is part of the acrosomal matrix material, and is not located specifically on the inner or the outer acrosomal membrane; (3) the matrix material plays an important mechanical role in stabilizing the position of the outer acrosomal membrane relative to the inner acrosomal membrane; it disperses during proacrosin activation.