By using the positional candidate gene approach, we were able to identify a novel serine protease gene that maps to chromosome 19q13.3-q13.4. Screening of expressed sequence tags allowed us to establish the expression of the gene and delineate its genomic organization (GenBank accession no. AF135023). We named this gene KLK-L1. Another group, by using a subtraction hybridization method, cloned the same gene and named it prostase (GenBank accession nos. AF113140 and AF113141). Here, we describe the precise mapping and localization of the prostase/KLK-L1 gene between the known genes KLK2 (human glandular kallikrein) and zyme (also known as protease M/neurosin). The direction of transcription of prostase/KLK-L1 is the same as that of zyme but opposite to that of KLK2 and prostate-specific antigen genes. Contrary to the initial impression, prostase/KLK-L1 is expressed at high levels not only in prostate tissue but also in testis, mammary gland, adrenals, uterus, thyroid, and salivary glands. We have further demonstrated with in vitro experiments with the breast carcinoma cell line BT-474 that this gene is expressed and that its expression is up-regulated by androgens and progestins. On the basis of information on other genes that are localized in the same region (prostate-specific antigen, KLK2, zyme, and normal epithelial cell specific-1 gene), we speculate that prostase/KLK-L1 may be involved in the pathogenesis and/or progression of prostate, breast, and possibly other malignancies.