Ca2+ is a universal intracellular messenger controlling a host of diverse physiological processes. Two housekeeping P-type Ca2+-transport ATPases, SERCA2b in the endoplasmic reticulum and SPCA1a in the Golgi, play a crucial role in cellular Ca2+ homeostasis by accumulating Ca2+ into their respective stores. SPCA1a also supplies the Golgi with Mn2+, a property that might be important for Mn2+removal/detoxification. Whereas SERCA2b contains two Ca2+-transport sites, SPCA1a possess only 1 Ca2+/Mn2+ site. No specific SPCA1a inhibitors are known, although SPCA1a inhibition might have therapeutic potential suppressing certain types of breast cancer. Some specific SERCA2b inhibitors have the ability to quench SPCA1a activity, although at higher IC50.Since endogenous expression levels of the ubiquitous SPCA1a are too low to perform extensive structure/function analysis, overexpression and purification strategies are inevitable. We successfully overexpressed and affinity purified recombinant SPCA1a from yeast. Purified pumps are then reconstituted in Golgi-like liposomes for detailed functional analysis. Reconstituted SPCA1a remains active displaying similar properties as expressed in mammalian fractions. The reconstituted system was now deployed to compare the potential of various compounds to inhibit SERCA and SPCA1a. Remarkably, the putative SPCA1a inhibitor Bisphenol displays equal affinity for SPCA1a and SERCA1a. Given the μM affinity of Thapsigargin (Tg) for SPCA1a and the fact that it can be chemically modified, Tg is used as a lead compound for generating more specific SPCA1a inhibitors. Several analogues were already tested. Furthermore, the purified and reconstituted SPCA1a is currently used to investigate the role of its N- and C-terminus, since the N- and C-termini of the related SPCA2 isoform interact with Orai1, a process known as store-independent Ca2+-entry that is activated in lactation and breast cancer.