A specific peptide inhibitor of the cyclic AMP (cAMP)-dependent protein kinase (PKI-peptide) is a very effective inhibitor of the cAMP-dependent activation of motility of Ciona spermatozoa, when PKI-peptide is present at the beginning of incubation of demembranated spermatozoa with cAMP and ATP. Under conditions where approximately 120 sec is required for full activation of motility, the window of sensitivity to the PKI-peptide lasts for only 25-30 sec. Examination of sperm pellet proteins labeled with 32P ATP during activation reveals a major 25 kDa phosphoprotein and 2 minor phosphoproteins whose phosphorylation is highly sensitive to to inhibition by the PKI-peptide and essentially complete during this early phase. These sperm proteins appear to be immediate substrates for cAMP-dependent protein kinase, and phosphorylation of one or more of these appears to be requires, but not sufficient, for activation of motility. The phosphorylation of other proteins is reduced or eliminated when PKI-peptide is present at the beginning of incubation, but is unaffected by later addition of PKI-peptide. Some of these substrates appear to be likely candidates for axonemal proteins that must be phosphorylated during the later stages of incubation in order to complete the activation process. This selection is based upon a high degree of inhibition by inclusion of PKI-peptide or other inhibitors at the start of the incubation process, on near-completion of their phosphorylation by the end of the 2 min incubation period required for the activation of motility, and evidence that these proteins are phosphorylated during in vivo activation of motility. Although these observations suggest the presence of a second kinase activity that is upregulated by the initial activation of the cAMP-dependent protein kinase, assays using exogenous substrates have not yet been able to identify such a kinase activity.