Demembranated and membrane disrupted bull sperm models exhibit an increase in motility when exposed to cAMP. Tritium-labeled cAMP was used to locate the initial site of action of cAMP in the modeled sperm preparations. cAMP did not bind selectively to the modeled cells, and the presence or absence of plasma membrane fragments on the models did not significantly alter this result. When suspension medium taken from modeled sperm preparations was subjected to gel filtration on Sephadex G25-150 columns, cAMP bound to a high molecular weight component that eluted with the void volume. The responsible binding factor is a soluble component that is released when the plasma membranes of the sperm are disrupted during the modeling procedure. To test the importance of the cAMP binding factor, modeled sperm were centrifuged, the supernatant solution was decanted, and the cells were resuspended in fresh medium. After this treatment the cells could be restored to motility with Mg-ATP but no longer exhibited a response to cAMP. Furthermore, addition of cAMP binding factor isolated by gel filtration partially restored the response of these sperm to cAMP. Investigation of the properties of the cAMP-binding factor have confirmed that it is specific for cAMP, with a much lower affinity for AMP and cGMP. In the presence of a large excess of unlabeled cAMP the labeled complex has a half-life of approximately 1 hour. Our results indicate that the action of cAMP on the motility of modeled sperm is mediated by its attachment to a high molecular weight, soluble component of the cell cytoplasm.