The pharmacokinetics and bioavailability of neomycin in sheep were investigated following intravenous, intramuscular, subcutaneous and intratracheal administration of 10 mg/kg. A rapid distribution phase (t 1/2 alpha, 3.16 min) was followed by a slower elimination phase (t1/2 beta, 1.98 h). The apparent volume of distribution was 304.69 ml/kg. Absorption half-lives were 18.62, 21.22 and 40.83 min and elimination half-lives 2.68, 2.82 and 2.55 h after IM, SC and IT administration, respectively. Bioavailabilities between 0.74 and 0.85 were obtained for the three routes of administration. Based on the bioavailability and disposition kinetics of neomycin, a twice daily IM dosage regimen should both be practical and adequate to maintain plasma neomycin concentrations within the pharmacologically active but nontoxic range.