The recent approvals of Photofrin-sensitized photodynamic therapy (PDT) in Canada and the Netherlands are significant events in the history of this relatively new cancer therapy. These developments will encourage further research in the design, synthesis, and evaluation of new photo- sensitizing agents, which seek to overcome certain limitations of the first generation prophyrins. Among these generally recognized limitations are weak absorption at wavelengths where optical penetration in tissue is optimal, relatively poor specificity with respect to tumor and normal tissue uptake and retention, and prolonged skin photosensitivity. A number of candidate 'second generation' compounds have been proposed and studied. The focus of these proposed studies is a family of cationic dyes that show particular promise as sensitizers of direct tumor cell destruction for PDT. Initial experience with the use of these compounds, described as Nile Blue derivatives, raises interesting and challenging research problems that are critical to resolve in order to optimize the effectiveness of these photosensitizers. Furthermore, these questions are likely to be of general importance in the search for optimum PDT methods of targeting tumor cells directly. On the basis of a body of experimental and theoretical work carries out in our laboratory over the past several years, we hypothesize that problems associated with photochemical oxygen consumption and diffusion will be critical aspects of optimizing the use of the Nile Blue derivatives for PDT. These issues are particularly important for these dyes in that they are bleached through enzymatic and photoreduction mechanisms under conditions of low oxygen concentration. Careful attention to the details of these processes can create significant opportunities for improved therapeutic efficacy. Towards this end, the application poses fives specific aims: (1) direct microelectrode measurements of PDT-induced oxygen consumption in Nile blue-sensitized multicell tumor spheroids and determination of photodynamic rates of oxygen consumption; (2) determination of the threshold dose for these dyes in the multicell tumor spheroid system; (3) optical sectioning (confocal) fluorescence microscopy of Nile blue-sensitized spheroids under various incubation conditions and during irradiation to determine bleaching rates and mechanisms; (4 optical reflectance spectroscopy of tumor oxygenation during Nile blue-sensitized PDT with emphasis on hemoglobin O2 saturation and cytochrome redox status; and (5) optimization of Nile blue-sensitized PDT irradiation protocols in three rodent tumor systems.