Fluorescence detection may constitute an appropriate means in gastrointestinal cancers to diagnose lymphatic tumor spread as opposed to gamma-scintillation methods. Photodiagnostic tracers have been shown to localize rapidly in malignant cells and may enable sensitive detection of small cell aggregates in lymph nodes. To reach a detection depth of several millimeters, a broad banded unspecific tissue autofluorescence may be controlled by so-called background subtracting techniques, generally based either on fluorescence observation at several wavelengths or on dual-wavelength fluorescence excitation. Using such comparative fluorescence detection techniques, some tumor entities can be differentiated soley based upon autofluorescence characteristics. Introducing a further enhancement in sensitivity for longer life-time fluorophores by time delayed fluorescence detection we ran a pilot trial comprising 174 lymph nodes from colorectal cancer specimen from 9 patients. Metastatically involved lymph nodes could be differentiated from all other palpable nodes in the mesenteric fat at a specificity of 85% with a sensitivity of 65%. Specific fluorescence features may be useful to preselect tissue samples for further histological analysis.