Aims To investigate the combined effect of hydrostatic pressure (HP), moderate temperature and nisin on the inactivation of Bacillus sporothermodurans spores which are known to be contaminant of dairy products and to be extremely heat-resistant. Methods and Results A central composite experimental design with three factors, using response surface methodology, was used. By analysing the response surfaces and their corresponding contour plots, an interesting interaction with the three factors was observed. The inactivation observed was shown to be well fitted with values predicted by the quadratic equation, since the adjusted determination coefficient (Radj2) was 0 center dot 979. The optimum process parameters for a 5-log sporesml-1 reduction of B.sporothermodurans spores were obtained, 472MPa/53 degrees C for 5min in presence of 121UIml-1 of nisin. Conclusion Nisin and temperature treatments improve the effectiveness of pressure in the inactivation of highly heat-resistant spores of B.sporothermodurans. Significance and Impact of the Study This study shows the potential of using high HP for a short time (5min) in combination with moderate temperature and nisin to inactivate B.sporothermodurans spores in milk. Such treatments could be applied by the dairy industry to ensure the commercial sterility of UHT milk.