A novel setup for fluorescence measurements of surfaces of biological samples, in particular the plasma membrane of living cells, is described. The method is based on splitting of a laser beam and multiple total internal reflections ͑TIR͒ within the bottom of a microtiter plate ͑cell substrate͒, such that up to 96 individual samples are illuminated simultaneously by an evanescent electromagnetic field. Main prerequisites are an appropriate thickness and a high transmission of the glass bottom,

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... hich is attached to the 96-well cell culture plate by a noncytotoxic adhesive. Glass rods of rectangular cross sections are optically coupled to this bottom for TIR illumination. Fluorescence arising from the plasma membrane of living cells is detected simultaneously from all samples using an integrating charge-coupled device ͑CCD͒ camera. The TIR fluorescence reader is validated using cultivated cells incubated with different fluorescent markers, as well as stably transfected cells expressing a fluorescent membraneassociating protein. In addition, particularly with regard to potential pharmaceutical applications, the kinetics of the intracellular translocation of a fluorescent protein kinase c fusion protein upon stimulation of the cells is determined.