Hyaluronidase is a hyaluronic acid-degrading endoglycosidase that is present in many toxins and the levels of which are elevated in cancer. Increased concentration of HYAL1-type hyaluronidase correlates with tumor progression and is a marker for grade (G) 2 or 3 bladder cancer. Using bladder tissues and cells, prostate cancer cells, and kidney tissues and performing reverse transcription-PCR, cDNA cloning, DNA sequencing, and in vitro translation, we identified splice variants of HYAL1 and HYAL3. HYAL1v1 variant lacks a 30-amino acid (aa) sequence (301-330) present in HYAL1 protein. HYAL1v1, HYAL1v2 (aa 183-435 present in HYAL1 wild type), HYAL1v3 (aa 1-207), HYAL1v4 (aa 260-435), and HYAL1v5 (aa 340-435) are enzymatically inactive and are expressed in normal tissues/cells and G1 bladder tumor tissues. However, HYAL1 wild type is expressed in G2/G3 tumors and in invasive tumor cells. Stable transfection and HYAL1v1-specific antibody confirmed that the HYAL1 sequence from aa 301 to 330 is critical for hyaluronidase activity. All tumor cells and tissues mainly express HYAL3 variants. HYAL3v1 lacks a 30-aa sequence (299-328) present in HYAL3 protein, that is homologous to the 30-aa HYAL1 sequence. HYAL3v1, HYAL3v2 (aa 251-417 present in HYAL3 wild type), and HYAL3v3 (aa 251-417, but lacking aa 299-328), are enzymatically inactive. Although splicing of a single independent exon generates HYAL1v1 and HYAL3v1, internal exon splicing generates the other HYAL1/HYAL3 variants. These results demonstrate that alternative mRNA splicing controls cellular expression of enzymatically active hyaluronidase and may explain the elevated hyaluronidase levels in bladder/prostate cancer.