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Distinguishing between motor and sensory fascicles is essential to successful peripheral nerve reconstruction. Acetyltiocholine staining methods based on the Karnovsky procedure have been increasingly used for the purpose. Our aim was to reduce the staining time, which is at least 45 minutes, so that the method could be used intraoperatively. We studied influence of staining time, temperature and components of staining solution on the results of staining. An additional oxidation step was introduced. The new method was evaluated by blind examinations of 5 differently stained groups of 30 randomized specimens of mixed and sensory rat nerves. Optimum parameters of the staining process were defined. The new modification with the added oxidation step had the same sensitivity and specificity rates as the more time-consuming standard methods and yielded sharper staining in only 30 minutes. The added oxidation step significantly reduces the time of motor and sensory fibre differentiation, and is suitable for clinical use.