Nanoparticle cytotoxicity testing based on in vitro methods frequently lack consistency. Even the inclusion of the commonly employed growth supplement, FCS (fetal calf serum), generates variable results. Thus, our object was to investigate the effect of FCS concentration on the cytotoxic behaviour of the unmodified nanoclay, Cloisite® Na+. Human monocytic U937 cells in medium supplemented with 5% FCS, 2.5% FCS or serum-free medium were treated with 1 mg/ml Cloisite Na+. Cell growth in 2.5% FCS was significantly inhibited by Cloisite Na+ within 48 h, whereas little effect was seen with a supplement of 5% FCS. Without serum, cell growth was inhibited and Cloisite Na+ had a detrimental effect on these cells. In media supplemented with FCS, the nanoclays agglomerated together to form large bundles, whereas they were evenly dispersed throughout the medium in the absence of serum. Clay particles, therefore, have cytotoxic properties that may be linked to their dispersion pattern. These adverse effects seem to be masked by 5% FCS. Serum supplementation is an important consideration in the toxicological assessments of nanomaterials on cells, which needs to be addressed in the standardization of in vitro testing methods.