Little has been reported on serum levels attained using once-daily aminoglycoside regimens and their relation to dosage administered and renal function. Consecutive patients with serious infections were randomized to receive gentamicin 4 mg/kg q 24h i.v. (n = 69), gentamicin 1.33 mg/kg q 8h i.v. (n = 46) or netilmicin 5.5 mg/kg q 24h i.v. (n = 59) (with dose reduction in case of renal dysfunction). In the three groups, median first serum trough levels were 0.4, 1.0 and 0.4 mg/l, respectively, and median first serum peak levels were 9.5, 4.7 and 12.2 mg/l (p < 0.01 once-daily vs. thrice-daily regimens). Dose adjustment because of first trough concentrations of > 2 mg/l and/or peak concentrations of < 6 mg/l was required in 6%, 78% and 12% of patients, respectively. Second trough and peak concentrations were significantly higher in the thrice-daily gentamicin group; serum levels remained constant in the other two groups. The six patients in the once-daily groups who developed elevated trough levels later in therapy were characterized in most cases by a decline in renal function.