OBJECTIVE To determine the incidence of bacterial colonization of intravascular catheters, to compare the incidence of colonization of intra-arterial (IA), intravenous (IV) and central venous catheters (CVC), and to determine the association, if any, between catheter withdrawal and bacterial sepsis. METHODS A prospective observational study was carried out at the neonatal intensive care unit of a university-affiliated regional referral centre. A total of 155 catheters (45 IA, 54 IV and 56 CVC) were obtained from 96 infants admitted to the unit and the distal 0.75 cm studied under the scanning electron microscope. The adjoining 0.75 cm was cultured for bacteria. RESULTS Scanning electron microscopy revealed that 46% of catheters had bacteria on the internal surface and 13% had bacteria on the outer surface. Greater numbers of CVC were colonized with bacteria compared to IA and IV catheters (P < 0.01). Bacterial colonization of intravascular catheters was not significantly associated with the duration the catheter remained in situ or local reaction at the site of entry of the catheter. Colonization of the external surface of the catheter was significantly associated with bacterial septicaemia (P = 0.0466). Eighty-three per cent of 155 catheters studied had coagulum on the inner or outer surface. Only 53% of these were colonized with bacteria. Bacterial colonization occurred in the absence of a coagulum in only three instances. Catheter withdrawal was not associated with bacterial sepsis. Lack of coagulum on the internal surface of the catheter was strongly associated with septicaemia during the 7 days after catheter withdrawal. CONCLUSIONS Although significant numbers of intravascular catheters were colonized with bacteria, only colonization with the external surface was associated with catheter-related sepsis.