Especially for animal protection reasons, tissue concentrations of intracisternally administered antibiotics in the mammary gland hardly can be determined in the live cow. Therefore, this paper assessed the use of the isolated perfused bovine udder to study the distribution of penicillins in glandular tissue. With this intention, the in vitro results acquired with this model were compared to tissue concentrations as well as absorption data from the few in vivo studies in the literature and differences were interpreted. This approach must consider inevitable deviations of experimental materials and methods. Furthermore, in vivo the udder is included in a closed circulatory system with other metabolism and excretion feasibilities than the isolated model. Moreover, the lower flow rate in the vessels in vitro has to be taken into account with respect to absorption capacities. Nevertheless, the tissue concentrations and the distribution equilibrium across the blood-udder-barrier in both experimental concepts corresponded with each other, if the deviating conditions are considered by calculating correction factors. Advantages and disadvantages of the isolated udder are discussed critically. In conclusion, this method is a useful completion of pharmacokinetic in vivo studies that are supposed to compare intracisternally administered formulations.