The sodium- and potassium-transporting ionophore monensin induces the maturation of Caenorhabditis elegans spermatids to spermatozoa in vitro. Rearrangement of cytoplasm, fusion of membranous organelles with the plasma membrane and growth of pseudopodia, all characteristic of in vivo spermiogenesis, occur within five minutes after exposure to monensin at concentrations of 0.1-1.0 micronM. This activation is dependent upon external Na+ and K+ ions but not Ca2+ ions. Monensin-activated spermatozoa have normal morphology and normal amoeboid motility. During activation spermatids twitch and rotate prior to pseudopod extension. Analysis of intermediates by transmission and scanning electron microscopy reveals that the sequence of morphogenetic events leading from the spherical spermatid to the polarized spermatozoan involves microvilli rearrangement and membranous organelle fusion, cytoplasmic polarization, then pseudopod extension.