Bovine mammary gland morphogenesis and differentiation are regulated by actions of growth factors including members of the transforming growth factor β superfamily. Activins A and B, which are members of the transforming growth factor β superfamily, bind selectively to ActRIB and ActRIIA receptors and their biological effects are antagonized by inhibins and follistatins. In the present paper we evaluated gene and protein expression of the activin and inhibin subunits βA, βB, and α-inhibin and follistatin and ActRIB and ActRIIA receptors in the mammary gland of nonpregnant and pregnant heifers. Mammary glands were obtained from nonpregnant Nelore (Bos indicus) heifers (n=9) and from primigravid Nelore heifers during early (n=9), mid (n=6), and late (n=5) pregnancy. Specimens of mammary tissue were analyzed by real-time PCR and immunohistochemistry. The βA and α-inhibin subunits and ActRIB and ActRIIA mRNA expression was higher in the early-pregnancy group compared with the nonpregnant group. In the mid-pregnancy group, the subunits βA, βB, and α-inhibin as much as follistatin mRNA expression was higher compared with the nonpregnant group, whereas ActRIB transcripts were absent in the late-pregnancy group. Immunostaining of these proteins, with the exception of ActRIB, was observed in the mammary tissue sections at all time points analyzed; these findings are in agreement with the observed pattern of mRNA expression. Staining and mRNA expression for ActRIB were undetected in the late-pregnancy group. In summary, the present study demonstrated that the activin-related proteins, βA, βB, and α-inhibin subunits, as much as follistatin and ActRIB and ActRIIA receptors display different patterns of expression regarding time of gestation in the bovine mammary gland. The modulation of the expression pattern during gestation suggests that activin-related proteins may play a key role in regulating bovine mammary branching morphogenesis and epithelial differentiation.