Although the jelly layers surrounding amphibian eggs are known to be essential to fertilization, no biological activities have been reported for jelly macromolecules in any vertebrate. Here we provide evidence that the jelly surrounding Xenopus laevis eggs releases a small diffusible protein into the surrounding media that serve as sperm chemoattractant. Using video microscopy we find that Xenopus sperm will preferentially turn toward and contact a glass capillary filled with egg jelly extract. In experiments using a two-chamber bioassay device we find that egg jelly extracts are capable of stimulating sperm movement across a membrane barrier sixfold over controls. This activity is not observed in materials unrelated to egg jelly and the response of sperm to egg jelly extract is clearly chemotactic rather than chemokinetic. A concentration gradient of the extract is absolutely necessary, and the chemotactic activity of the extract exhibits a biphasic dose dependence similar to that of chemotactic agents in other systems. We have been able to characterize the factor as being a heat-stable protein about 10 kDa in size. This study, therefore, provides the first clear evidence for a diffusible sperm chemoattractant in a nonhuman vertebrate, as well as the first demonstration of a physiological role for egg jelly macromolecules in Xenopus fertilization.