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Catheter-related infections pose a hazard to both humans and laboratory animals. The aim of this study was to develop a technique preventing bacterial colonization of intravascular catheters. In 27 dogs a total of 70 catheters were implanted. On an average catheters were used for 207 days. Three protocols were compared: (1) flushing the catheters with a heparinized solution; (2) filling only the catheter lumen with alpha-chymotrypsin solution (225 units/ml); (3) filling only the catheter lumen with a solution containing a mixture of the aminoglycoside antibiotic gentamicin (20 mg/ml) and chymotrypsin (225 units/ml). Catheter fillings were always withdrawn before catheter use. Catheter exit sites were all treated with povidone iodine ointment once a day. Body temperatures and weights were recorded, bacteriological and electron microscopical examinations of catheters performed. Without gentamicin filling all catheters were colonized after a few weeks. The dogs showed clinical signs of chronic bacteraemia. Gentamicin filling eradicated colonization. No further bacteraemia was observed. We conclude that filling only the catheter lumen with a concentrated solution of chymotrypsin and gentamicin, combined with measures to prevent infections via the subcutaneous catheter tunnel, is an effective and safe technique to prevent catheter-related infections.