Apoptosis, a form of noninflammatory cell death, plays a central role in mammary gland involution after weaning. Previous studies have shown that apoptosis in the post- weaning mammary gland is substantially reduced by treat- ment with glucocorticoids or progesterone, but whether these steroids exert a similar antiapoptotic effect during normal lactation is not known. Therefore, the present study used an in vivo rat model to assess the effects of progesterone and glucocorticoids on apoptosis in the lactating mammary gland. Rats were untreated, sham operated, ovariectomized (OVX), and/or adrenalectomized (ADX) on d 10 of lactation. Additional groups of OVX/ADX rats were treated with either progesterone or corticosterone. Mammary gland apoptosis was determined 3 d later by 3-end labeling of fragmented DNA and by in situ terminal deoxynucleotidyl transferase- mediated deoxyuridine triphosphate biotin nick end label- ing analysis (TUNEL). DNA fragmentation was relatively low in the mammary gland from untreated and sham-operated rats and was unaffected by either ADX or OVX alone. In contrast, DNA fragmentation was markedly elevated in OVX/ADX rats (P < 0.01), but this effect on mammary gland apoptosis was prevented by replacement with either corticosterone or proges- terone. Consistent with these data, dying cells identified by TUNEL analysis were readily observed in the alveolar epithe- lium of mammary tissue from OVX/ADX rats but not in any of the other groups. These data demonstrate that during normal lac- tation, mammary gland apoptosis is inhibited by endogenous progesterone and glucocorticoids. Importantly, the presence of either steroid alone was sufficient to prevent apoptosis, sug- gesting that their antiapoptotic effects in the lactating mam- mary gland may be mediated via similar signaling pathways. (Endocrinology 143: 222-227, 2002)