The study of spermatogenic cell physiology has been hindered by the absence of unbiased methods of identification of cells upon which single cell techniques are being applied. In this work, we have used histochemical techniques, digital videoimaging, quantification of chromatin-bound DNA probes, and measurements of cell diameter to identify single spermatogenic cells at different periods of development. Our criteria of identification permit the definition of four developmental stages of spermatogenesis on which to perform single cell analyses: spermatogonia B/preleptotene spermatocytes, leptotene/zygotene spermatocytes, pachytene spermatocytes, and round spermatids. The use of voltage-sensitive dyes and Ca(2+)-sensitive dyes does not interfere with the estimations of DNA content. The estimations of DNA content of spermatogenic cells can be performed both with near-UV excited dyes (H33342) and long wavelength-excited dyes (ethidium bromide), allowing the use of a wide range of physiological and immunocytochemical fluorescent probes to study the spermatogenic process.