Hypericin is a polycyclic, aromatic, naphthodianthrone which has been shown to possess in vivo and in vitro antiretroviral activity. To gain further insight into the mechanism(s) by which hypericin exerts its antiretroviral effects, we have studied Radiation Leukemia virus (RadLV) produced from cells pulse-treated with hypericin. Hypericin-treatment did not inhibit retroviral production or the proteolytic cleavage of the gag-encoded precursor proteins. Rather, hypericin was found to be associated with RadLV particles, the retrovirions showed an increased density in sucrose, and the RadLV protein banding patterns were altered. RadLV produced from hypericin-treated cells was rendered noninfectious upon exposure to visible light. Our results suggest that RadLV produced from hypericin-treated cells is inactivated by a hypericin-mediated photodynamic process.