Two samples of highly pure multiwalled carbon nanotubes (MWCNTs) similar in hydrophobicity and surface reactivity were prepared with similar length, <5 μm, but markedly different diameter (9.4 vs 70 nm). The samples were compared for their cytotoxic activity, uptake, and ability to induce oxidative stress (ROS production and intracellular GSH depletion) in vitro in murine alveolar macrophages (MH-S). The in vivo toxicity was evaluated by measuring biochemical (LDH activity and total proteins) and cellular responses in bronchoalveolar lavage (BAL) after intratracheal instillation in rats. Both samples were internalized in MH-S cells. However, thin MWCNTs appeared significantly more toxic than the thicker ones, both in vitro and in vivo, when compared on a mass-dose basis. The data reported herein suggest that the nanotube diameter is an important parameter to be considered in the toxicological assessment of CNTs.