Antibiotic concentrations were determined in the lacrimal fluid of sheep following subcutaneous application of penicillin/ dihydrostreptomycin into the lower eyelid, and intramuscular administration of spiramycin base, tiamulin, and oxytetracycline formulations. The penetration of penicillin and dihydrostreptomycin into the lacrimal fluid was poor. The spiramycin and tiamulin concentrations in the lacrimal fluid were 10‐ and 4‐fold higher than in the serum. The peak spiramycin concentration in the lacrimal fluid was 3.4 ±0.8 μg/ml at 8 h post injection (p.i.) and the drug could be detected at least 72 h p. i. For tiamulin and oxytetracycline (OTC) peak concentrations of 1.53 ±0.70 and 1.88 ±1.9 μg/ml, respectively, were achieved in the lacrimal fluid and these drugs could be detected 25 to 30 h p.i. The OTC and tiamulin concentration‐time curves for lacrimal fluid and serum were parallel, whereas for the spiramycin appearance in the lacrimal fluid was delayed.