INTRODUCTION: Gold nanoparticles (AuNPs) have unique properties that promise new and improved methods for targeting cancer treatment and diagnosis. However, despite their relatively high biocompatibility, AuNPs can negatively affect cell viability. Research indicates that the interactions with the plasma membrane and cellular uptake of AuNPs depend significantly on size, shape, and surface modifications. MATERIAL AND METHODS: We evaluated the use of human lymphocyte primary culture as a model for assessing the to-xicity of AuNPs in proliferating cells. We compared the toxicity of rod-shaped, PEGylated AuNPs (gold nanorods, AuNRs) of two different sizes and gold nanospheres (AuNSs). RESULTS: Our results show that at high concentrations, both AuNSs and AuNRs negatively affect the viability of activated human lymphocytes . The cytotoxic effect varies with size and concentration, with larger AuNRs (approx. 22 × 50 nm) being more toxic than smaller ones (approx. 20 × 40 nm) and 15 nm AuNSs exhibiting the lowest toxicity. CONCLUSIONS: Our results confirm that the application of AuNPs in cancer the-rapy and diagnostics must be accompanied by a thorough cytotoxicity assessment. Despite certain limitations, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction test for viability assessment of proliferating cells proves to be a simple and cost-effective method useful in nanoparticle toxicity studies.