The absorption, distribution and excretion of netilmicin (NTL) in rats were examined by the microbiological assay method. Plasma level and urinary excretion of NTL in rats after intramuscular administration were compared with those of 5 other aminoglycoside antibiotics, gentamicin, sisomicin, dibekacin, amikacin and tobramycin. 1. After intravenous administration, plasma level of NTL declined rapidly with a half-life of about 18 minutes. In the cases of intramuscular, intraperitoneal and subcutaneous administrations, plasma levels reached maximum at 15 minutes and declined with similar half-life as intravenous administration. 2. When 20 mg/kg of NTL was administered intramuscularly, NTL was highly distributed into the kidney, while poorly into the brain. The disappearance rates of NTL from the tissues except the kidney were almost same as that from the plasma. Concentration of NTL in kidney was about 3 times higher than that in plasma at 15 minutes after administration and declined with a half-life of about 7 days. 3. NTL excreted in the bile within 24 hours was only 0.2% of the dose administered (20 mg/kg, i.m.). An average concentration of bile during the first 2 hours was 3 mcg/ml. 4. Irrespective of the route of administration (i.v., i.m., i.p. and s.c.), NTL was excreted rapidly in the urine and 70.0 approximately 81.0% of the dose was recovered within 6 hours. 5. Plasma level and urinary excretion of NTL after intramuscular administration (20 mg/kg) were almost similar to those of 5 other aminoglycoside antibiotics. 6. There was no difference between NTL and GM in plasma level after high dose (100 mg/kg, i.m).