Targeting cytotoxic 4β-phorbol esters toward cancer tissue was attempted by conjugating a 4β-pborbol derivative with substrates for the proteases prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) expressed in cancer tissue. The hydrophilic peptide moiety was hypothesized to prevent penetration of the prodrugs into cells and prevent interaction with PKC. Cleavage of the peptide in cancer tumors was envisioned to release lipophilic cytotoxins, which subsequently penetrate into cancer cells. The 4β-phorbol esters were prepared from 4β-phorbol isolated from Croton tiglium seeds, while the peptides were prepared by solid-phase synthesis. Cellular assays revealed activation of PKC by the prodrugs and efficient killing of both peptidase positive as well as peptidase negative cells. Consequently no selectivity for enzyme expressing cells was found. KEYWORDS: 4β-Phorbol ester, protease-assisted targeting, targeted chemotherapy, prodrug, prostate-specific antigen. prostate-specific membrane antigen P rostate cancer (PCa) is a major cause of death by cancer in men in high-income countries. 1 In the initial stage, PCa mainly consists of cells that are androgen-dependent, and the growth can be retarded by hormone therapy. 2 Unfortunately, in later stages hormone refractory cells dominate (castrationresistant prostate cancer, CRPC). 2,3 At this stage the use of common chemotherapeutics is complicated by the slow proliferation of the cancer tissue, since chemotherapeutics like taxanes, doxorubicine, or vincristine target the proliferative stages of cancer. Thus, selectivity is obtained by the faster division rate for cancer cells. 3−6 Therefore, an urgent need for drugs against late-stage PCa exists.Preclinical evidence supports the idea that drugs targeting protein kinase C (PKC) may be useful in treatment of CRPC. 7 The PKC family comprises ten serine/threonine kinases, which can be divided into three groups: (i) conventional PKC (cPKCs: -α, -βI, -βII, and -γ), (ii) novel PKCs (nPKCs: -δ, -ε, -θ, and -η), and (iii) atypical PKCs (aPKCs: -ζ, -ι, and λ). Expression and function of different PKC isoforms are context-