To understand the mechanism regulating flagellar bending in spermatozoa, it is important to investigate the regulation of microtubule sliding in the flagellar axoneme. It has been shown that protease treatment following demembranation with Triton X-100 disrupts the connections between microtubules and induces extrusion of microtubules from the flagellar axoneme. This approach enables a direct investigation of the regulation of microtubule sliding; however, the percentage of spermatozoa with protease-induced extrusion was relatively low, probably due to protease digestion of some regulatory motility proteins, as well as proteins connecting the microtubules. In this study, we demonstrate microtubule extrusion in most hamster and mouse demembranated spermatozoa upon treatment with a high concentration of the reducing agents dithiothreitol or 2-mercaptoethanol, without the use of proteases. The extrusion of microtubules occurred when the spermatozoa were treated with concentrations of the reducing agents that were sufficient for the reduction of the disulfide bonds of IgG. These results suggest that the arrangement of the axonemal structures connecting doublet microtubules depends to an important degree on -S-S- bonds. Close observation of the extrusion process using the present method revealed that microtubules were extruded on the same side as that of the curve of the sperm head, and also on the opposite side. Furthermore, we noted that extrusion always started on one side, followed by the other side, but was never initiated on both sides simultaneously.