The transcellular Ca2+ fluxes required for milk production must be rigorously regulated to maintain the low cytosolic Ca2+ concentrations critical to cell function. Ca2+-ATPases play a critical role in the maintenance of this cellular Ca2+ homeostasis. Using RT-PCR and sequencing, we identified six Ca2+ pumps in lactating mammary tissue. Three plasma membrane Ca2+-ATPases (PMCAs) were found (PMCA1b, PMCA2b, and PMCA4b). Two sarco (endo)plasmic reticulum Ca2+-ATPases (SERCAs) were identified (SERCA2 and SERCA3), and the rat homologue to the yeast Golgi Ca2+-ATPase RS-10 was also found. The pattern of mRNA expression of each of these pumps was examined in rat mammary tissue from the 7th day of pregnancy to the 21st day of lactation. Northern blots revealed increased mRNA expression for all Ca2+ pumps by the 14th day of lactation, and transcripts continued to increase through the 18th day of lactation. PMCA1b, PMCA4b, SERCA2, and SERCA3 showed the lowest levels of expression. RS-10 transcripts were more abundant than SERCA2, SERCA3, PMCA1b, and PMCA4b. RS-10 was the only pump to increase in expression before parturition. PMCA2b was the most abundant transcript found in lactating mammary tissue. At peak lactation, expression of PMCA2b approached that of actin. The high expression, high affinity for Ca2+, and high activity at low calmodulin concentrations exhibited by PMCA2b suggest that it is uniquely suited for maintenance of Ca2+ homeostasis in the lactating mammary gland. The pattern of expression and abundance of RS-10 suggest that it is a candidate for the Golgi Ca2+-ATPase shown to be important in maintaining the Golgi Ca2+ concentration required for casein synthesis and micelle formation.