MGEA5 was originally identified to be a novel human hyaluronidase, which is immunogenic in meningioma patients. Recently an N-acetylglucosaminidase was reported with identical sequence. Here, we define the origin of a splice variant by determining the genomic organization of the mgea5 gene. We find the splice variant missing a putative acetyltransferase domain of MGEA5. As for evolutionary analysis, we show that the MGEA5 is highly conserved in higher eukaryotes, As for expression analysis, we find both mRNA variants ubiquitously expressed in various human tissues and throughout mouse development, We generated polyclonal antibodies against MGEA5s/5 and identified proteins of 75 and 130 kDa, indicating posttranslational modifications of the larger protein. Cell fractionation revealed the cytoplasmic/cytoskeletal localization of the 130-kDa protein and the nuclear localization of the 75-kDa protein. We propose a model in which MGEA5 functions both as a hyaluronidase and an N-acetylglucosaminidase.