A study was conducted to evaluate the effects of feeding salinomycin at the recommended prophylactic level, and at 2 and 3 times this level, to finishing male broilers (d 21 to 38). Four treatment groups were given the experimental diets containing 0, 60, 120, or 180 parts per million (ppm) salinomycin from d 21 to 38. Performance, relative organ weights, selected serum enzymes, and salinomycin residues in liver, muscle, and serum were determined. Salinomycin supplementation had no effect on body weight, feed intake, or feed conversion, and caused no overt signs of toxicity. After a week of being fed the salinomycin diets, the serum activity of aspartate aminotransferase was significantly increased in chickens fed 180 ppm compared with controls. These birds also showed microscopic lesions in breast and thigh muscles, but not in cardiac muscle. Salinomycin residues were not detected by high-performance liquid chromatography coupled to tandem mass spectrometry in liver or muscle samples from the birds fed 0, 60, or 120 ppm salinomycin. However, chickens fed 180 ppm salinomycin had detectable levels in liver and muscle above the maximum residue level of 5 μg/kg established by the European Union. All birds fed salinomycin had salinomycin in their sera with levels ranging from N.D. (not detected) in the controls to 24.4 ± 7.9, 61.4 ± 18.9, and 94.5 ± 9.1 μg/L for salinomycin dietary levels of 60, 120, and 180 ppm, respectively. Serum salinomycin concentration was linearly related with salinomycin content in feed (y = 0.584x - 10, r2 = 0.999). The results showed that even at 3 times the prophylactic level, salinomycin does not induce clinical toxicosis or mortality. No salinomycin residues were found in edible tissues at the recommended dietary level or at 2 times this level. However, salinomycin was detected in serum regardless of the dietary level. A simple method for salinomycin determination in serum is described which can be used as a marker of exposure and/or to predict levels in the diet.