The unique properties of carbon nanotubes (CNTs) have been explored for their use in biomedical sciences and in biotechnological fields; however, their possible toxic effects are of concern. The sources of human exposure to nanomaterials include inhalation, ingestion, dermal contact and injection. The pulmonary and dermal effects of CNTs in vitro have been previously studied with contradictory results, but data on intestinal cells are scarce. The aim of this study was to evaluate the cytotoxicity of single wall CNTs functionalized with carboxylic acid on differentiated and non-differentiated Caco-2 cells, derived from a human intestinal adenocarcinoma. Biomarkers assessed were neutral red uptake (NR), protein content (PT), a tetrazolium salt (MTS) metabolization, LDH leakage (LDH) and cell viability by means of the trypan blue exclusion test (TBET). Moreover, a morphological study was performed. Cells were exposed to concentrations between 5 and 1,000 microg/ml CNTs and toxic effects were studied after 24h of exposure. NR and MTS results showed a concentration-dependent trend with an inhibitory response from 100 microg/ml CNT, together with an increase in LDH leakage. TBET resulted in an 80% reduction at higher concentrations, and finally PT was only modified at higher concentrations. Overall, results indicated cytotoxic effects on the Caco-2 cells with differentiated cultures showing a higher sensitivity. Thus, a hazard assessment of CNTs is necessary as the nanotechnology industry grows, and more nanoscale wastes are released into the environment.