Background P-type ATPases are ubiquitous ion and lipid pumps found in cellular membranes. P5A-ATPases constitute a poorly characterized subfamily of P-type ATPases present in all eukaryotic organisms but for which a transported substrate remains to be identified. Scope of review This review aims to discuss the available evidence which could lead to identification of possible substrates of P5A-ATPases. Major conclusions The complex phenotypes resulting from the loss of P5A-ATPases in model organisms can be explained by a role of the P5A-ATPase in the endoplasmic reticulum (ER), where loss of function leads to broad and unspecific phenotypes related to the impairment of basic ER functions such as protein folding and processing. Genetic interactions in Saccharomyces cerevisiae point to a role of the endogenous P5A-ATPase Spf1p in separation of charges in the ER, in sterol metabolism, and in insertion of tail-anchored proteins in the ER membrane. A role for P5A-ATPases in vesicle formation would explain why sterol transport and distribution are affected in knock out cells, which in turn has a negative impact on the spontaneous insertion of tail-anchored proteins. It would also explain why secretory proteins destined for the Golgi and the cell wall have difficulties in reaching their final destination. Cations and phospholipids could both be transported substrates of P5A-ATPases and as each carry charges, transport of either might explain why a charge difference arises across the ER membrane. General significance Identification of the substrate of P5A-ATPases would throw light on an important general process in the ER that is still not fully understood. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.