In many mammals, sperm are immotile while stored in the caudal epididymis and do not become motile until ejaculation. We report here our investigation of the mechanism that initiates motility in mature rat epididymal sperm. We found that an external "activator" is not required to initiate rat sperm motility since immotile sperm started to swim immediately when exposed to solutions that contributed only osmotic support. Instead, we found that epididymal rat sperm are kept fully immobilized by a high molecular weight glycoprotein, "immobilin," that we have isolated from rat cauda epididymal (CE) fluid. Our results strongly support the hypothesis that immobilin inhibits sperm motility mechanically simply by creating a highly viscoelastic environment: 1) rat CE fluid inhibited the motility of such disparate cells as rat sperm, E. coli. and rabbit sperm (which are fully motile in rabbit CE fluid), 2) the degree to which a variety of enzymatic treatments or slight dilution of the CE fluid initiated sperm motility depended only on the degree to which the treatment reduced the viscoelastic drag of the fluid, and 3) centrifugation of CE fluid simultaneously copurified the component of the fluid that immobilizes the sperm, the component that renders the fluid viscoelastic, and the glycoprotein immobilin.