Objectives: The aim of this randomized study was to investigate the effects of once versus twice daily gentamicin dosing on renal function and measures of infectious disease in a population with infective endocarditis (IE). Methods: Seventy-one IE patients needing gentamicin treatment according to guidelines were randomized to either once (n = 37) or twice daily (n = 34) doses of gentamicin. Kidney function (glomerular filtration rate, GFR) was measured with an isotope method ( 51Cr-EDTA) at the beginning of treatment and at discharge. Treatment efficacy was assessed by C-reactive protein (CRP) time to half-life, mean CRP and leukocytes. Results: Baseline GFR was similar in the two groups. Both groups displayed a significant fall in GFR from admission to discharge. The mean decrease in GFR was as follows: with once daily gentamicin, 17.0% (95% confidence interval 7.5-26.5), and with twice daily gentamicin, 20.4% (95% confidence interval 12.0-28.8). However, there was no significant difference in the GFR decrease between the once and twice daily regimens (p = 0.573). No difference in infection parameters was demonstrated between the two dosing regimens. Conclusions: A twice daily gentamicin dosing regimen is neither less nephrotoxic nor more efficient than a once daily regimen in the treatment of IE patients. When indicated, gentamicin may therefore also be administered as a single-dose regimen in the treatment of IE patients.