To assess the histological features of in vivo toluidine blue (TB) uptake in potentially malignant oral lesions (PML) and to determine whether any were related to clinical Dark versus Pale Royal Blue stain and/or to the malignant/dysplastic nature of the lesions. Frozen sections were used to evaluate TB extra- and intra-epithelial distribution, depth of penetration and nuclear or extra-nuclear uptake. Eighteen lesions were studied. The clinical appearance of a Dark Royal Blue stain was significantly related to the nuclear uptake of the dye. Conversely Pale Royal Blue staining was unrelated to any histological feature. Dark Royal Blue-malignant lesions had more nuclear uptake than benign lesions. The results suggest that Dark Royal Blue staining is the true positive outcome of a TB test and showed that Dark Royal Blue-malignant and Dark Royal Blue-benign lesions have a different histological pattern of uptake.