Bovine mammary involution, an important process for subsequent lactations, is characterized by loss of epithelial cells by apoptosis, but its hormonal regulation is still not well defined. Prolactin (PRL) and growth hormone (GH) play a specific role on rat mammary gland apoptosis, through insulin-like growth factor 1 (IGF-1) and the IGF binding protein (IGFBP) system. The purpose of our investigation was to determine the possible role of PRL, GH, and IGF-1 on cell survival and on IGFBP-5 expression in the bovine mammary gland. Mammary gland explants were cultured in the presence of cortisol, 17beta-estradiol, progesterone, insulin, PRL, GH, and IGF-1 and with the same treatment but without PRL, GH or IGF-1, respectively. After 24 h of culture, we determined the level of apoptosis through evaluation of DNA laddering in the oligonucleosomal fraction and examined IGFBP-5 messenger RNA (mRNA) expression. The results show a high level of DNA laddering and an increase in IGFBP-5 mRNA content in mammary explants cultured in the absence of PRL, GH, or IGF-I with respect to explants treated with all hormones. Moreover, explants cultured in presence of PRL, GH, or IGF-I show a low level of DNA laddering and IGFBP-5 expression with respect to explants cultured without any hormones. These data demonstrate a relationship between levels of apoptosis and IGFBP-5 mRNA expression in the bovine mammary gland and confirm the involvement of this binding protein programmed cell death and its relationship with the main lactogenic hormones.