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Mammalian spermatogenesis is a complex process which is not yet fully understood. In this paper we describe the analysis of rat spermatogenesis by means of 3‐parameter flow cytometry. Since the analysis of DNA content only provides sufficient information for the identification of 4 cell populations, additional parameters were combined with propidium iodide (PI) staining. Immunostaining of the intermediate filament vimentin allowed the identification of somatic (vimentin positive) and germ (vimentin negative) cells. Utilizing the combination of DNA and vimentin staining, we have been able to quantitate the somatic cells present in a testicular cell suspension and to analyze somatic and germinal cells separately. Furthermore, the addition of mitochondrial staining with the fluorochrome nonyl acridine orange (NAO) allowed several cell subpopulations within each ploidy group to be distinguished. After 3‐color staining and subsequent cell sorting, 11 testicular cell subpopulations could be identified: somatic cells, and 10 subtypes of germinal cells. The method described in this paper represents a valuable tool for the evaluation of spermatogenesis in both normal and perturbed situations. Cytometry 27:161–168, 1997. © 1997 Wiley‐Liss, Inc.

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