Few reports have addressed neonatal rifampicin plasma concentrations and data on neonatal rifampicin pharmacokinetics are completely lacking. Therefore, plasma concentrations of rifampicin and its main metabolite 25-O-desacetylrifampicin (DES) were measured in 123 surplus plasma samples from routine vancomycin monitoring in 21 neonates using reversed-phase HPLC. Rifampicin peak and trough plasma concentrations were 4.66 +/- 1.47 mg/L and 0.21 +/- 0.20 mg/L, respectively, after a dose of 8.5 +/- 2.1 (mean +/- SD) mg/kg per day. A significant linear relationship between rifampicin dose and peak plasma concentrations was found, but inter-patient variability was high. Pharmacokinetic parameters of rifampicin were calculated according to a one-compartment open model with iterative two-stage Bayesian fitting (MW\PHARM 3.60, Mediware, The Netherlands). First-order elimination constant, volume of distribution corrected for weight, total body clearance corrected for weight (CL/W), and elimination half-life were 0.16 +/- 0.06 h(-1), 1.84 +/- 0.59 L/kg, 0.28 +/- 0.11 Lkg(-1) h(-1), and 4.9 +/- 1.7 h, respectively. A high Pearson correlation was found between CL/W rifampicin and the covariates plasma creatinine and CL/W gentamicin of a preceding gentamicin treatment course, r = 0.728 (n = 17) and r = 0.837 (n = 12), respectively. DES was detected in each plasma sample. Therefore, rifampicin seems to be eliminated by both renal and metabolic pathways in neonates. In 8 study patients, plasma concentrations of rifampicin and DES were measured again after two weeks of therapy. CL/W rifampicin was significantly higher (67 +/- 50%). The authors suggest maintaining the current dose regimen of 10 mg/kg once a day. Because of the large inter-patient variability in rifampicin plasma concentrations and CL/W increase during therapy, the authors suggest monitoring rifampicin peak and trough plasma concentrations to avoid low plasma concentrations. More research is needed to determine well-founded dosing guidelines.