Intracellular development of microsporidian parasites comprises a proliferative phase (merogony) followed by a differentiation phase (sporogony) leading to the release of resistant spores. Sporogony implies, successively, meront-to-sporont transformation, sporont division into sporoblasts, and sporogenesis. We report a procedure improving the separation of sporogonial stages of Encephalitozoon cuniculi, a species that develops inside parasitophorous vacuoles of mammalian cells. Supernatants of E. cuniculi-infected Madin-Darby canine kidney cell cultures provided a large number of parasites mixed with host-cell debris. This material was gently homogenized in phosphate-buffered saline containing 0.05% saponin and 0.05% Triton X-100 then filtered through glass wool columns. Centrifugation of the filtrate on 70% Percoll-0.23 M sucrose gradient gave a reproducible pattern of bands at different densities. Transmission electron microscopy showed that three of the four collected fractions were free of visible contaminants. Corresponding prominent cell stages were early sporoblasts (fraction B), late sporoblasts plus immature spores (fraction C), and mature spores (fraction D). Further centrifugation of the lightest fraction (A) on 30% Percoll-0.23 M sucrose gradient generated a sporont-rich fraction (A2). First analysis of proteins from fractions A2 and D by two-dimensional gel electrophoresis suggested a potential use of the described method for proteomic profiling.