Protein phosphorylation appears to be a necessary step in the intracellular signaling pathway that initiates the activation of sperm motility. Activation of live immotile sea urchin sperm produced rapid, time-dependent increased phosphorylation on proteins of 32, 45, 130, and 500 kDa. Fractionation of immotile and motile sperm indicated that these motility-related phosphoproteins are associated with flagella. These proteins showed greater phosphorylation in the flagellar fraction from motile sperm, suggesting that subcellular boundaries are in place to keep protein kinases and their substrates spatially separated. Solubility properties suggest that these proteins are the heavy chain and smaller subunits of sea urchin sperm dynein which are phosphorylated in vivo to initiate activation of motility. This also suggests that phosphorylation of only these few proteins, out of the nearly 100 phosphorylations known to occur in the basic axoneme, appears to be associated with the early signaling pathways of motility activation in intact sperm.