novel approach is proposed for fluorescence imaging of biological substrates. Discrimination among the contributions of different fluorophores is achieved in the time domain, taking advantage of the differences in decay times of exogenous versus endogenous fluorescences. The experimental setup relies on an intensified video camera and on a subnanosecond dye laser used for the excitation. The video camera has an electronic shutter that provides exposure times as short as 5 ns. A detailed description of the apparatus is reported with an analysis of performances. The time-gated technique is especially attractive for tumor detection in combination with photosensitizing drugs. Experiments performed on a murine tumor have shown interesting results that make this technique promising for a future application in the clinical diagnosis.