Mammalian spermatozoa acquire the capacity to be motile during passage through the epididymis; spermatozoa removed from the testis or caput epididymis and placed in a buffered saline solution are generally nonmotile, whereas spermatozoa removed from the cauda epididymis or vas deferens and placed in the same solution display high motility. One of the most powerful tools for analysis of the regulation of spermatozoan movement is the demembranated, reactivated model. These are spermatozoa deprived of their plasma membrane by treatment with a nonionic detergent, and then reactivated or induced to beat in a solution containing MgATP2- so that the effects of various ions and substances on their axonemal movement can be examined directly. Lindemann and Gibbons first achieved the reactivation of mammalian (bull and human) spermatozoa using a modification of the method developed by Gibbons and Gibbons for reactivation of sea urchin spermatozoa; the reactivated bull spermatozoa exhibited beat frequencies and waveforms very similar to those of intact spermatozoa. This chapter describes procedures for the demembranation and reactivation of mature golden hamster and ram spermatozoa that results in beating of the flagella of virtually 100% of the demembranated spermatozoa with a waveform closely resembling that of the intact spermatozoa. It also describes procedures for the collection and reactivation of immature golden hamster spermatozoa.