Animals show a faster clearance and a lower incidence of nephrotoxicity and ototoxicity when aminoglycosides are administered during the activity period. Human data on a circadian rhythm in pharmacokinetics are conflicting, and there are no data on a circadian rhythm in toxicity. When aminoglycosides are administered once daily, as is often done, a circadian rhythm in pharmacokinetics or toxicity could have clinical implications. In a prospective study we investigated the influence of drug administration time on serum drug levels and the incidence of nephrotoxicity in 221 patients with serious infections treated with gentamicin or tobramycin once daily. We did not find statistically significant differences in trough or peak levels for the three time periods (midnight to 7:30 AM, 8 AM to 3:30 PM, and 4 to 11:30 PM). Nephrotoxicity occurred significantly more frequently when the aminoglycosides were administered during the rest period (midnight to 7:30 AM; p = 0.004). In addition to the coadministration of high-dose furosemide or other nephrotoxic antibiotics and the duration of treatment, the time of administration was still an independent risk factor in a multivariate analysis.