In the amoeboid spermatozoa from Caenorhabditis elegans, motility acquisition is preceded by substantial rearrangement of the plasma membrane. The current genetic model posits a multicomponent complex of membrane and cytoplasmic proteins responsible for pseudopod extension. This model can be translated into a physiological context through the involvement of cholesterol-enriched signaling platforms. We show that discrete cholesterol-enriched microdomains are present in C. elegans spermatids. These microdomains redistributed towards the cell body upon pseudopod extension resulting in a phospholipid-enriched pseudopod. Cholesterol saturation in the spermatids prevented pseudopod extension and motility acquisition, whereas cholesterol depletion increased the rate of in vitro pseudopod extension. This work suggests that plasma membrane cholesterol plays an important role in regulating the membrane dynamics that precede pseudopod extension and motility acquisition. (C) 2012 Elsevier Inc. All rights reserved.