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Targeting cancer vasculature is an emerging field in cancer treatment. Photochemical internalization (PCI) is a drug delivery technology based on photochemical lysis of drug-bearing endocytic vesicles originally designed to target cancer cells. Recent investigations have revealed a lower PCI efficacy in vascular endothelial cells (HUVECs) in vitro than in HT1080 fibrosarcoma cells. This manuscript aims to explore the limiting factor for the PCI effect in HUVECs. Cellular uptake of the photosensitizers AlPcS(2a) and TPPS(2a), and a model compound for macromolecular drugs taken up by fluid phase endocytosis, Alexa⁴⁸⁸-dextran, was explored by flow cytometry. The uptake of AlPcS(2a) and TPPS(2a) was 3.8-fold and 37-fold higher in HUVECs than in HT1080 cells, respectively, while the Alexa⁴⁸⁸-dextran uptake was 50% lower. AlPcS(2a) (but not TPPS(2a)) was shown to reduce Alexa⁴⁸⁸-dextran uptake in a concentration-dependent manner, resulting in 66% and 33% attenuation of Alexa⁴⁸⁸-dextran uptake at 20 μg/ml AlPcS(2a) in HUVECs and HT1080 cells respectively. Studies of intracellular localization of Alexa⁴⁸⁸-dextran and AlPcS(2a) by confocal microscopy in HUVECs uncovered a concentration-dependent AlPcS(2a)-induced inhibition of Alexa⁴⁸⁸-dextran trafficking into AlPcS(2a)-stained and acidic vesicles. The localization of Alexa⁴⁸⁸-dextran to AlPcS(2a)-localizing compartments was reduced by 40% when the AlPcS(2a) concentration was increased from 5 to 20 μg/ml. The treatment dose of AlPcS(2a) was found to influence on the efficacy of PCI of saporin, but to a lesser extent than expected considering the data from cellular uptake and intracellular trafficking of Alexa⁴⁸⁸-dextran. The implications of these results for further development of vascular targeting-PCI are discussed.