Placental transfer and excretion into milk of 14C-isepamicin sulfate (14C-HAPA-B) following a single intramuscular or intravenous dose of 25 mg/kg were studied in pregnant or lactating rats. Concentrations of HAPA-B in placenta, ovary and uterus reached their maxima in 10 minutes after administration then declined rapidly. The maximum concentration in the fetal membrane was similar to 10-minute levels in these other tissues, but was attained in 4 hours or later after the drug administration and some drug still remained there even at 24 hours. A small amount of radioactivity was distributed into the fetus and the maximum level in the fetus was attained in 1 approximately 4 hours after administration, much later than in maternal tissues. The concentration in the fetal kidney was the highest in the fetus, but only 1 microgram/g or lower was found. A very small amount of radioactivity was also found in the fetal bone by radioautography. The drug was excreted into milk at 2 approximately 4 micrograms/ml during the first 6 hours and decreased a little in 24 hours after administration. There was no difference in results due to administration routes.