A subset of cancer stem cells termed 'side population' (SP) cells has been identified in several types of solid tumor and is known to be responsible for the failure of chemotherapy and for cancer relapse. In the present study, the oral squamous cell carcinoma (OSCC) cell line SCC‑55 was used for analysis of the SP cells based on Hoechst 33342 exclusion, via the ATP‑binding cassette (ABC) transporter, with and without verapamil using fluorescence activated cell sorting. The results demonstrated that the presence of SP cells in the SCC‑55 cell line was 2.8%, which was reduced to 0.6% in the presence of verapamil, an inhibitor of the ABC transporter. In addition, following exposure to the chemotherapeutic drug 5‑fluorouracil, a high survival rate was observed in the SP cells due to overexpression of the ABCG2 and B‑cell lymphoma 2 proteins. In addition, overexpression of stem cell surface markers, including CD44 and CD147 was observed in the SP cells. Therefore, these findings suggested that OSCC contains SP cells, which share certain characteristics with stem cells. The coexpression of ABC transporters and stem cell surface markers in SP cells may be associated with resistance to chemotherapeutic agents and also supports a role for these cells in tumor recurrence, metastasis and invasion.