Serotonin (5-HT) functions as a feedback inhibitor of lactation. This action is mediated by 5-HT receptors (5-HTR). In situ hybridization studies showed 5-HTR subtypes are expressed in bovine mammary epithelial and myoepithelial cells as well as blood vessels, and vary by receptor subtype. The aim of this study was to determine the distribution of the 5-HTR subtypes (1B, 1E, 2A, 2B, 4 and 7) in cattle, goat and sheep mammary glands using immunohistochemistry, and to compare distribution among lactating and dry udders. Hypothalamus and mammary gland samples were taken at slaughter from 3 lactating and 3 nonlactating animals of each species. Hypothalamal tissue was used as positive control. Tissues were fixed, paraffin embedded and 5-µm sections were placed on slides. Immunohistochemical staining was performed using rabbit primary antibodies against 5-HTR for the 1B, 1E, 2A, 2B, 4 and 7 subtypes. Anti-rabbit secondary antibody was conjugated with streptavidin peroxidase, and visualization of binding was realized using diaminobenzidine substrate. Tissues were counterstained with hematoxylin. Myoepithelial cells were identified in serial sections by positive immunohistochemical staining for calponin. All 6 receptor subtypes were expressed in mammary epithelial cells in the 3 species studied. Furthermore, 5-HTR 1E was expressed in the myoepithelial cells in all 3 species, and in the blood vessels of cows. Receptor subtype 4 was found in myoepithelial cells of cows and sheep. In all 3 species blood vessels were positively stained for receptors 1B, 2A and 2B. In the lactating animals, receptor distribution in the mammary epithelial cells was cytoplasmatic. However, in mammary tissue from nonlactating animals, distribution changed to the apical membrane in all species. In conclusion, 5-HTR are expressed in epithelial, myoepithelial and blood vessels within mammary tissue of cattle, sheep and goats, with distribution of subtype varying by cell-type and species. Furthermore, distribution of the receptors on mammary epithelial cells changes between lactating and nonlactating states.