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Morphology of the crayfish spermatozoon and of the spermatophore wall during three stages of final maturation including freshly ejaculated, post-mating, and after spermatozoa release was studied and compared. The crayfish spermatophore consists of a sperm mass enveloped by a three layered spermatophore wall. After mating, the thickness of the outer layer of the spermatophore is increased. The matrix in the middle layer of the spermatophore becomes reticulated, and granules inside this layer release their contents. Fibers in the inner layer degrade to small particles. The spermatozoon capsule swells and the space between the capsule and the spermatozoon appears. The area of the plasma membrane is increased by wrinkling of the surface and alteration from a single to a multilayered structure at the anterior part of the acrosome. The density of the subacrosome zone increases in the vicinity of the main body of the acrosome. With the onset of fertilization, the layers of the spermatophore are dissolved by female glair gland secretions. The spermatozoon extracellular capsule, plasma membrane, and membranous lamellae are eliminated, and bundles of filaments are released from anterior part of the acrosome. The subacrosome zone loses electron density and retracts. The electron-dense material of the innermost layer of the acrosome is discharged and, together with acrosome filaments, forms a filament/droplet structure at the anterior part of the spermatozoon. The most important change is observed in the subacrosome zone, which may play a key role in the fertilization. Also, morphological changes of the spermatozoon that occur after release from the capsule, especially formation of the filament/droplet structure, may contribute to the mechanism of egg-spermatozoon binding in the crayfish, representative of animals with non-motile spermatozoa., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)