Sixty percent of calcium in milk is transported across the mammary cells apical membrane by the plasma membrane Ca2+-ATPase 2 (PMCA2). The effect of abrupt cessation of milk production on the Ca2+-ATPases and mammary calcium transport is unknown. We found that 24 h after stopping milk production, PMCA2 and secretory pathway Ca2+-ATPases 1 and 2 (SPCA1 and 2) expression decreased 80-95%. PMCA4 and Sarco/Endoplasmic Reticulum Ca2+-ATPase 2 (SERCA2) expression increased with the loss of PMCA2, SPCA1, and SPCA2 but did not increase until 72-96 h of involution. The rapid loss of these Ca2+-ATPases occurs at a time of high mammary tissue calcium. These results suggest that the abrupt loss of Ca2+-ATPases, required by the mammary gland to regulate the large amount Of Calcium associated with milk production, could lead to accumulation of cell calcium, mitochondria Ca2+ overload, calcium mediated cell death and thus play a part in early signaling of mammary involution. Published by Elsevier Inc.