19 preterm and 2 newborn infants received gentamicin in dosages of 2.5 mg/kg every 12 h. Successive determinations of serum concentrations show, that the levels of gentamicin are adequate for therapy even in preterm infants with low birth weight. Concentrations 30 min after injection (c30min) and base-line-concentrations vary widely. 74% of the 30 min-serum levels are observed within the desired therapeutic range, between 3 and 10 microgram/ml. 16% of the levels measured 30 min after intravenous or intraarterial injection are higher than the known range for potential ototoxicity of 10 microgram/ml. 10% of the 30 min-levels are around 2 microgram/ml, so that the therapeutic efficacy during the following interval of application is doubtful. There is no evidence of accumulation of the drug for periods of treatment up to 7--20 days. The average serum half-life in premature infants with a gestational age of less than 32 weeks and a birth-weight of less than 2000 g is 5--6,7 h. Those with a gestational age of greater than 32 weeks and a birth-weight of greater than 2000 g, and full-term infants show a gentamicin half-life of 2.9h. To determine the actual serum concentration of gentamicin, the rapid and easy disc-agarose-diffusion test using B. subtilis is useful and suitable for routine therapy control.