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We previously reported that vancomycin in hyperalimentation solution reduces catheter-related infections in the neonatal intensive care unit. Since June 1993 vancomycin (25 μg/ml) was routinely added to central venous catheter solutions, primarily hyperalimentation solution. Because the prophylactic use of vancomycin could lead to the emergence of resistant organisms, the decision to discontinue this practice was made in April of 1999. The use of vancomycin was reserved for documented infections with vancomycin-susceptible organisms.To compare catheter longevity, rate of laboratory-confirmed blood stream infections and total vancomycin exposure between two 18-month periods before and after the cessation of prophylactic vancomycin use.Data were evaluated for every neonate in whom a percutaneous central venous catheter was placed.There were 394 neonates enrolled. No statistically significant difference was identified between the two periods regarding the mean catheter days or number of catheters per patient. There was a higher rate of Gram-negative laboratory-confirmed blood stream infections during Period I in patients with percutaneous central venous catheters in place. There were more isolates of coagulase-negative staphylococci in Period II, resulting in more frequent vancomycin therapy institution and thus an overall increase in the amount of vancomycin used in that periodDiscontinuing the use of prophylactic vancomycin resulted in exposure of fewer neonates to vancomycin but a higher total amount of vancomycin used. The impact of low dose widespread exposure to vancomycin vs. high dose limited exposure on the microbiologic flora in the neonatal intensive care unit should be further examined.