Calcium pumps and channels modulate cell proliferation and apopto- sis by regulating intracellular calcium (Ca2þ). The plasma membrane Ca2þ ATPase isoform, PMCA2, is a calcium efflux mechanism that extrudes Ca2þ from the cytosol into the extracellular space. PMCA2 has a restricted expression, including expression in cochlear hair cells and cerebellar Purkinje cells. PMCA2 expression is increased in mouse mammary glands during lactation where it plays a major role in the excretion of Ca2þ into milk; however, PMCA2 expression has not been assessed in human breast tissue exhibiting lactational changes. Our previous studies have shown that PMCA2 mRNA levels are elevated in some breast cancer cell lines and that pan-PMCA antisense attenuates the proliferation of MCF-7 breast cancer cells. However, the consequences of silencing PMCA2 in breast cancer cells are still not well understood. Our study assessed PMCA2 expression in breast tissue exhibiting lactational change and in human malignant breast tissue samples. The role of PMCA2 in the proliferation of breast cancer cells was also evaluated. Immunohis- tochemistry using a rabbit anti-PMCA2 antibody showed membranous PMCA2 expression in the luminal epithelium of breast tissue exhibiting lactational change. PMCA2 expression was assessed in human breast tumor samples assembled into tissue microarrays. Nine of 96 breast tumours (9.4%) showed membranous PMCA2 staining. PMCA2 expression did not significantly correlate with the breast cancer pathological mar- kers, estrogen, progesterone or HER2 receptor status. High-content imaging demonstrated that PMCA2 silencing in MDA-MB-231 breast cancer cells is associated with a reduction in cell number and an inhibition of the percentage of S-phase positive cells. The effect of PMCA2 silencing combined with various cytotoxics (cisplatin, doxorubicin or mitomycin C) on cell proliferation was assessed in MDA-MB-231 cells using a kinetic imaging system (IncuCyte). The results showed that PMCA2 silencing promotes the effects of some cytotoxics. These findings indicate that PMCA2 protein expression is elevated during human lacta- tion and in some breast cancers. Inhibitors of PMCA2 may represent a novel therapeutic strategy for some breast cancers.