We have developed a large-scale purification method of the phosphorylated form (131 kDa) of membrane-bound guanylyl cyclase (mGC) from Hemicentrotus pulcherrimus spermatozoa. The purified mGC contained 26.0 ± 1.3 moles of phosphate/mol enzyme (mean ± S.D., n = 6). Phosphorylated peptides were isolated from the trypsin digest of the carboxymethylated H. pulcherrimus sperm mGC by affinity chromatography on a Chelating Sepharose Fast Flow column, and the peptides were then subjected to mass spectrometric analysis and determination of phosphoserines, after the conversion of phosphoserines to Sethylcysteines by amino acid analysis. Based on the observed mass number and the content of phosphoserine, serine residues at positions 561, 565, 652, 722, 740, 755, 894, 897, 914, 918, 927, 930, 951, and 985, in addition to two residues among those at positions 666, 670, and 671, were shown to be phosphorylated. They are all located in the intracellular region (kinase-like and catalytic domains). Notably, serine residues at positions 894, 918, 927, and 930, that are conserved in the sequence of mammalian mGCs and medaka fish-eye-specific mGCs, are phosphorylated in the sea urchin sperm mGC.