The aim of this study was to assess the concentration of penicillin G in mammary tissue and secretion of dry heifers following systemic administration of penethamate hydriodide. Six dairy heifers in late gestation received a single intramuscular injection of 10 g penethamate hydriodide and were sacrificed 24, 48 or 144 h after treatment. Penicillin G concentrations were measured in mammary tissue and secretion samples using HPLC. Penicillin G was detected in the udder of two animals euthanized at 24 h (mammary tissue and secretion) and at 48 h post treatment (mammary secretion only) after administration at concentrations still close to or above MIC90 values reported for the pathogens associated with heifer mastitis. Antibiotic concentration shortly after administration will have been substantially higher indicating a potential for systemic treatment with penethamate hydriodide to control prepartum intramammary infections in heifers without the disadvantages of local therapy such as teat contamination or risk of trauma for the administrator.