Since in mouse spermatozoa the somatic histones are replaced by other basic proteins and there are changes in the chromatin supraorganization, different patterns of extended chromatin fiber (ECF) formation would be expected compared with those formed by somatic cells that were previously studied. In this study, we investigated the formation of ECF in mouse testicular spermatozoa after lysis with 2 M NaCl plus 1% Triton X-100, and under the action of gravity. ECFs were observed under polarized light in fixed and unfixed spermatozoa subjected to lysis in a vertical position and stained with toluidine blue at pH 4.0. In unfixed preparations, all of the sperm nuclei showed ECFs, whereas in fixed preparations 60% of the cells had ECF. The latter frequency was much higher than that previously reported for mouse hepatocytes. Even in cells that did not produce ECFs in vertically and horizontally lysed preparations, an ordered reorganization of the chromatin was observed after lysis. The faint positive response to acid fast green at the nuclear periphery in spermatozoa that did not develop ECF after lysis was assumed to represent residual protamine and nuclear matrix proteins. The high frequency of mouse sperm cell nuclei with ECF probably reflected the extraction of protamines from the DNA-protein complexes of sperm cell nuclei facilitated by the specific lysis protocol.