Abstract Background and purpose: Thorough knowledge of the RBE of clinical proton beams is indispensable for exploiting their full ballistic advantage. Therefore, the RBE of the 200-MeV clinical proton beam produced at the National Accelerator Centre of Faure (South Africa) was measured at different critical points of the depth-dose distribution. Material and methods: RBEs were determined at the initial plateau of the unmodulated and modulated beam (depth in Perspex = 43.5 mm), and at the beginning, middle and end of a 7-cm spread-out Bragg peak (SOBP) (depths in Perspex = 144.5, 165.5 and 191.5 mm, respectively). The biological system was the regeneration of intestinal crypts in mice after irradiation with a single fraction. Results: Using 60Co γ-rays as the reference, the RBE values (for a γ-dose of 14.38 Gy corresponding to 10 regenerated crypts) were found equal to 1.16 ± 0.04, 1.10 ± 0.03, 1.18 ± 0.04, 1.12 ± 0.03 and 1.23 ± 0.03, respectively. At all depths, RBEs were found to increase slightly (about 4%) with decreasing dose, in the investigated dose range (12–17 Gy). No significant RBE variation with depth was observed, although RBEs in the SOBP were found to average a higher value (1.18 ± 0.06) than in the entrance plateau (1.13 ± 0.04). Conclusion: An RBE value slightly larger than the current value of 1.10 should be adopted for clinical application with a 200-MeV proton beam.