Gold nanoparticles (AuNPs) are relevant in nanomedicine for drug delivery in the vascular system, where endothelial cells are the first point of contact. We investigated the uptake of 80 nm AuNPs in primary human umbilical vein endothelial cells (HUVECs) by flow cytometry, 3D confocal microscopy, nano-scale 3D-imaging using focused ion beam/scanning electron microscopy (FIB/SEM), and single particle inductively coupled plasma-mass spectrometry (spICP-MS). HUVECs were cultured for 3 h or 24 h in medium with AuNPs in a concentration range of 1.25–10 μg ml−1. There was a concentration-dependent increase of AuNPs inside cells measured by flow cytometry, spICP-MS and 3D confocal microscopy. The latter also showed that AuNPs were located in the cytosol. This was supported by FIB/SEM, showing that AuNPs were located in membrane enclosures in the cytoplasm as single particles or agglomerates of 2–3 or more particles. Pre-treatment with chlorpromazine inhibited the AuNP-uptake in HUVECs, indicating that internalisation occurred mainly by clathrin-mediated endocytosis. Cell activation by exposure to tumour necrosis factor or lipopolysaccharide had a slight or no effect on the uptake of AuNPs, respectively. The AuNP exposure did not influence cell cytotoxicity, whereas the intracellular reactive oxygen species production was slightly increased. In conclusion, the uptake of AuNPs by endothelial cells can be addressed quantitatively by several methods with high throughput and/or high specificity. Uptake of AuNPs in HUVECs occurred mainly by clathrin-mediated endocytosis and trafficking to membrane enclosures in the form of single particles and agglomerates of 2–3 particles.