Prostate cancer (PC) is the most prevalent non-cutaneous cancer in men and leads to high morbidity and mortality. Disseminated PC is treated by hormonal treatment in order to block intratumoral activation of the androgen receptor (AR) by 5α-dihydrotestosterone (DHT). Despite this treatment, eventually all patients develop castration-resistant disease (CRPC) which is accompanied by poor prognosis. Novel androgen synthesis and receptor inhibitors improve patient survival in CRPC, demonstrating the crucial role of residual androgen presence in PC despite castration.Besides the classic DHT synthesis pathway through testosterone, recent studies in humans have shown formation of DHT through an alternative pathway that does not require testosterone as an intermediate. Activity of this alternative androgen synthesis pathway is dependent on the presence of specific steroidogenic enzymes. Results of preliminary preclinical and clinical research show that levels of alternative pathway intermediates are increased in CRPC.Our goal is to investigate the activity and relevance of this novel pathway during progression of PC. The contribution of the alternative pathway to DHT-initiated AR activation will be explored using previously established in vitro models and sensitive steroid detection methods. This translational study will serve to discover novel targets for inhibition of intratumoral DHT synthesis and subsequent (CR)PC growth.