Matriptase and its cognate inhibitor, HAI-1, have been implicated in carcinoma onset and malignant progression. However, pathologic mechanisms of matriptase activation are not defined. Steroid sex hormones play crucial roles in prostate and breast cancer. Therefore, we investigated the questions of if and how steroid sex hormones regulate matriptase activation in these cancer cells. Treatment of cells with 17-estradiol had no effect on activation of matriptase in hormone-starved breast cancer cells, in part due to their high constitutive level of activated matriptase. In striking contrast, very low levels of activated matriptase were detected in hormone-starved, LNCaP prostate cancer cells. Robust activation of matriptase was observed as early as 6 hr after exposure of these cells to 5-dihydrotestosterone (DHT). Activation of matriptase was closely followed by shedding of the activated matriptase with more than 90% of total activated matriptase present in the culture media 24 hr after DHT treatment. Activated matriptase was shed in a complex with HAI-1 and may result from simultaneously proteolytic cleavages of both membrane-bound proteins. Latent matriptase and free HAI-1 were also shed into culture media. As a result of the shedding, the cellular levels of matriptase and HAI-1 were significantly reduced 24 hr after exposure to DHT. DHT-induced matriptase activation and shedding were significantly inhibited by the androgen antagonist bicalutamide, by the RNA transcription inhibitor actinomycin D, and by the protein synthesis inhibitor cycloheximide. These results suggest that in LNCaP cells androgen induces matriptase activation via the androgen receptor, and requires transcription and protein synthesis.