Type II transmembrane serine proteases (TTSPs), including hepsin, are a new class of cell surface catalytic enzymes. In the present study, a non-transmembrane isoform of hepsin, named hepsin/-TM that originates from alternative splicing, was identified. Unlike the transmembrane hepsin isoform, this non-transmembrane isoform was distributed within the cytoplasm. Real-time PCR experiments revealed that while hepsin was expressed in all tested human tissues, hepsin/-TM was restricted in kidney, brain and lung tissues. Significantly, hepsin/-TM was not expressed in liver where hepsin was originally identified. However, hepsin/-TM was highly expressed in brain where hepsin was expressed at a relatively lower level. Moreover, these two isoforms showed different expression patterns in a number of colon adenocarcinoma cell lines. In addition, in contrast to hepsin, expression of hepsin/-TM in vivo does not exert any apparent inhibitory effect on mammalian cell growth.