Abstract Gentamicin is readily used for suspected or proven sepsis in neonates, yet it shows considerable inter-individual pharmacokinetic variability, which limits achievements of therapeutic levels. Hence, the aim of this study was to compare peak and trough gentamicin concentrations according to dosing regimen, to evaluate pharmacokinetic parameters, and to consider adjustments of dosing regimen. Babies with infection were treated with 1 h infusion, and daily dose of 5 or 7.5 mg/kg depending on the age. Patients were randomized into two groups: I — dosing interval 12 h (n=8), II — 24 h (n=11). Two steady-state blood samples were obtained. Pharmacokinetic parameters were calculated using one-compartment model. The results showed a difference (p<0.05) in peak gentamicin concentrations between the groups, and tendency of lower trough levels in the group II. Calculated pharmacokinetic parameters included the volume of distribution (Vd) 0.52±0.47 l/kg, clearance (CL) 0.055±0.036 l/hkg and a half-life (t1/2) of 6.89±3.21 h. Based on the method for dosing regimen adjustments, there was a need to extend dosing interval to 36 h in 6 patients