Seven protein phosphatases were isolated from extracts of spermatozoa and sperm tails of the sea urchin Hemicentrotus pulcherrimus by ion exchange and gel filtration chromatographies using [³²P]-histone and/or p-nitrophenyl phosphate (pNPP) as substrates and characterized for their enzymatic and molecular nature. Two of them isolated from the particulate fraction correspond to the mammalian type 1 and 2B protein phosphatases. Another two obtained from the soluble fraction were similar to the mammalian protein phosphatases, type 2A and 2C. A protein phosphatase corresponding to the mammalian type 1 enzyme (molecular mass of 43 kDa) dephosphorylated the [³²P]-autophosphorylated regulatory subunit of H. pulcherrimus sperm cAMP-dependent histone kinase.