Malignant transformation of mouse skin by chemical carcinogens and tumor promoters, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, is a multistage process leading to the formation of squamous cell carcinomas. In an effort to identify target genes whose expression is associated with skin tumorigenesis we combined elements of suppression subtractive hybridization with differential screening to isolate genes that are differentially upregulated in mouse skin after short-term treatment with 12-O-tetradecanoylphorbol-13-acetate and that exhibit a high constitutive expression in squamous cell carcinomas. Here, we report the detailed analysis of one of these cDNAs encoding the serine protease BSSP in mouse skin. Phorbol ester application increases BSSP expression in keratinocytes of the epidermis and the hair follicle several-fold starting 4 h post- treatment. Transcriptional activation of BSSP by 12-O-tetradecanoylphorbol-13-acetate was found to be independent of c-Fos expression and resistant to downregulation by glucocorticoids. By monitoring BSSP expression throughout experimental skin carcinogenesis we found strong constitutive expression in hyperplastic epidermis as well as in proliferatively active keratinocytes of benign and malignant skin tumors. These results establish a novel link between expression of an as yet ill-defined serine protease and skin carcinogenesis.