Inositol polyphosphate 4-phosphatases (IP4Ps) are enzymes involved in the regulation of phosphoinositide 3-kinase (PI3K) signaling. IP4Ps catalyze the hydrolysis of the D-4 position phosphoester of the PI3K generated lipid second messenger, phosphatidylinositol 3,4-bisphosphate. Western blot analysis detected the expression of a novel 110 kDa form of IP4P type Ia in mouse spleen, heart, lung, and uterus. In addition, the 110 kDa form of IP4P type la was found to be the major form of this enzyme expressed in human platelets, MEG-01 megakaryocytes and Jurkat T-cells. RT-PCR analysis of MEG-01 megakaryocytes and Jurkat T-cells indicates that the 110-kDa form of IP4P la is derived from an alternatively spliced mRNA that encodes an additional internal domain of 40 amino acids not present in the two previously described brain IP4P I alpha spliceoforms. The predicted molecular mass of this spliceoform is 109,968 Da, consistent with its apparent molecular mass estimated by Western blot analysis. The novel domain is proline rich and contains a PEST sequence characteristic of proteins that are rapidly degraded by the calpain family of proteases. Analysis of genomic DNA sequence indicates that the IP4P type I gene consists of 25 exons and that this novel spliceoform is obtained as a result of an unusual type of differential splicing involving the use of an alternative 5 ' -GU donor splice site during the excision of intron 15. In addition, we show that all three known spliceoforms of IP4P la result from alternative splicing involving exon 15 and 16 indicating that structural variability in this region of the enzyme may be important for its function.