Prostate-specific membrane antigen (PSMA) is a protein up-regulated in the vast majority of prostate cancers. Antibodies to PSMA have proved highly specific for prostate cancer cells, and the therapeutic potential of such antibodies is currently being assessed in clinical trials. We have previously shown that PSMA at the cell surface of polarized epithelial cells is predominantly expressed at the apical plasma membrane and that microtubule depolymerization abolishes apical PSMA targeting. In the current report, we implicate a functional role for a target membrane soluble N-ethylmaleimide-sensitive factor adaptor protein receptor, syntaxin 3, in the microtubule-dependent apical targeting of PSMA. PSMA and syntaxin 3 are similarly localized to the apical plasma membrane of the prostatic epithelium and Madin-Darby canine kidney cells. Introduction of a point mutation into syntaxin 3 abolishes its polarized distribution and causes PSMA to be targeted in a nonpolarized fashion. Additionally, treatment of polarized Madin-Darby canine kidney cells with vinblastine, a microtubule depolymerizing chemotherapeutic agent, causes both syntaxin 3 and PSMA to redistribute in a nonpolarized fashion. However, following treatment with the microtubule stabilizing chemotherapeutic agent Taxotere, both syntaxin 3 and PSMA continue to localize in a polarized manner at the apical plasma membrane. Thus, microtubule depolymerizing and stabilizing chemotherapeutic drugs might exact similar cytotoxic effects but have disparate effects on protein targeting. This phenomenon might have important clinical implication, especially related to antibody-mediated immunotherapy, and could potentially be exploited for therapeutic benefit.