An improved protocol for crystallographic refinement by simulated annealing is presented. It consists of slow cooling starting at high temperatures. Tests of refinements of aspartate aminotransferase and procin pepsin show that the slow-cooling protocol produces lower R factors and better geometry than other protocols previously published. The influence of the temperature-control method, weighting, cooling rate and duration of the heating stage on the success of the slow-cooling protocol is studied. Analysis of the time course of the potential-energy fluctuations indicates no global changes in the state of order of the system. Fluctuations of the potential energy are interpreted as localized conformational changes during the course of the refinement.