The major constitutive phosphatidylinositol-3,4,5-P3 (PtdIns) 5-phosphatase activity was purified and subjected to peptide sequence analysis providing extensive amino acid sequence which was subsequently used for cloning the cDNA. Peptide and cDNA sequences revealed that the purified PtdIns(3,4,5)P3 5-phosphatase was identical to a splice variant of a recently cloned inositol polyphosphate 5-phosphatase termed synaptojanin. Since synaptojanin is not known to possess PtdIns(3,4,5)P3 5-phosphatase activity, we verified that the purified PtdIns(3,4,5)P3 5-phosphatase activity and synaptojanin are identical by Western blot using specific antibodies raised against synaptojanin sequences. Immunoprecipitation from crude lysates of rat brain tissue showed that synaptojanin accounts for the major part of the active PtdIns(3, 4,5)P3 5-phosphatase activity. It is also shown that the protein is localized to the soluble fraction. Expression of a truncated recombinant protein demonstrates that the conserved 5-phosphatase region of the synaptojanin gene expresses PtdIns(3,4,5)P3 5-phosphatase activity. However, immunological analysis demonstrates that the PtdIns(3,4,5)P3 5-phosphatase activity expressed from the synaptojanin gene in brain is due to a particular splice variant which contains a 16-amino acid insert as shown by immunoprecipitation using a specific antibody raised against this particular splice variant.