The aim of this in-vitro study was to investigate the extent of autofluorescence in carious human dentine in comparison to the established caries diagnostic methods of microhardness testing, microradiography and histology. For this, 21 extracted, permanent human teeth with dentine lesions were chosen and embedded in methylmethacrylate. Then they were sectioned through the centre of the lesion, and approximately 150 µm thin slices were prepared parallel to the cut surface. The microhardness was tested on the block surfaces; the slices of the teeth were used to test the other investigative techniques. The fluorescence analysis was performed at an emission of over 515 nm due to 450 nm – 490 nm excitation and an emission of over 615 nm due to 530 nm – 585 nm excitation. The fluorescence scans corresponded to the mineral loss profiles as well as to the microhardness profiles and the carious expansion seen in the histological analysis. The differences between the two tested fluorescence filter systems were very small. The correlation between lesion depth, derived from microhardness testing and microradiography, and fluorescence plots were between 0.85 and 0.90. The results for histopathology were even better. It can be concluded from these outcomes that fluorescence can be used as a marker for carious dentine.