In the treatment of serious infection by aminoglycoside antibiotics multiple daily treatment with netilmicin is considered to be the least toxic. Studies comparing netilmicin with gentamicin using the less toxic once-daily schedule are lacking. A randomized prospective study was designed to evaluate the efficacy and toxicity of once-daily netilmicin with gentamicin treatment in patients with serious infections. Consecutive patients with serious infections were randomized between gentamicin 4 mg/kg q24h iv or netilmicin 5.5 mg/kg q24h iv. Exclusion criteria were neutropenia or severe renal failure. A good clinical response was observed in 50 of the 54 evaluable patients (92.6%) treated with gentamicin and in 48/52 (92.3%) netilmicin treated patients. Nephrotoxicity developed in 5/72 (6.9%) gentamicin patients and in 10/69 (14.5%) treated with netilmicin. Audiometry was performed with high-frequency audiometry when possible; no significant differences were found between the two aminoglycosides. We conclude that with once-daily treatment no benefit of netilmicin over gentamicin regarding nephro- or ototoxicity could be demonstrated.