The immunohistochemical reactivity of human, monkey, shrew, rat and mouse normal mammary glands was examined using methacarn-fixed paraffin-embedded specimens and acetone-fixed frozen sections using the avidin-biotin-peroxidase method for cell phenotype comparison. Actin was visualized using anti-smooth muscle actin antibody and keratin expression was determined by employing 12 different monoclonal antibodies. All these antibodies cross-reacted specifically with the species examined. Basal (myoepithelial) cells from all species showed muscle-specific actin according to reactivity with HHF35 monoclonal antibody. Keratin expression showed significant phenotypic differences among species. In human and monkey, AEL-KS2, KL1 CK8.13, AE3 and 34BE12 stained luminal cells as well as basal cells. AE1, RPN1165, CK4.62, 35BE11, M20 and RPN1162 labeled only luminal cells whereas 312C8-1 preferentially bound to basal cells. In shrews, AEL-KS2, CK8.13 and AE3 reacted to both cell types, AE1 reacted only with luminal cells, and 35BE12 and 312C8-1 selectively stained basal cells. In rodents, AEL-KS2 reacted to both cell types, CK8.13, AE3, 34BE12 and 312C8-1 stained rat basal cells, and 34BE12 and 312C8-1 reacted to mouse basal cells. The data represents cytoskeletal differences among species.