BACKGROUND: The SERS imaging of the intracellular microenvironment may provide valuable insight into the chemical composition and distribution of a cellular biological system. OBJECTIVE: In order to develop the SERS-based methodology for the cellular uptake of gold nanoparticles conjugated with the fluorescent dye (rhodamine 6G) was investigated in endothelial EA. hy. 926 cell line. A few timeframes were chosen to assess an effect of particles accessibility on their distribution in the cells. METHODS: Confocal Raman imaging was employed to evaluate the distribution of SERS response within the cells incubated with labelled Au nanoparticles. A cluster K-means analysis was used in the construction of SERS maps. RESULTS: The cellular uptake of the gold nanoparticles labelled with rhodamine 6G occured up to 12 h of the incubation. Along with the SERS signal of the dye, the bands originating mainly from aromatic amino acid residues and nucleobases were found in the spectra. CONCLUSIONS: The optimal accumulation of label-nanoparticles is found for up to 6 h incubation of the particles, confocal SERS imaging of labelled endothelial cells can serve as a powerful tool for further examination of the physiological and pathological changes in endothelial intracellular microenvironment.