Tilmicosin is a long-acting macrolide antibiotic currently approved for treatment of bovine respiratory disease in the USA. Serum pharmacokinetics of tilmicosin were compared between cattle (major species) and sheep (minor species) after subcutaneous injection in order to evaluate a new potential application of the drug in currently nonapproved species. There were no significant differences in the elimination rates, maximum serum concentrations, half-lives (t1/2), areas under the curve (AUC), areas under the first-moment curve (AUMC), and mean residence times. Volume of distribution (Vd(area)) and clearance (Cl), when normalized by body weight, were also similar. The only significantly different parameter was time at which maximum drug concentration was reached (tmax), with sheep having the tmax of 3.9 h, compared to 0.5 h in cattle. Although macrolides are considered to be one of the safest anti-infective drugs, adverse cardiovascular effects of several macrolides have been reported. The cardiopulmonary effects of tilmicosin were monitored in healthy adult sheep after receiving a single subcutaneous (s.c.) injection of tilmicosin at the dose of 10 mg/kg, and no significant changes were found. The study indicates that tilmicosin can be used safely and effectively in sheep at the given dose, with no adverse cardiopulmonary effects.