The naturally occurring sesquiterpene lactone thapsigargin is a potent and selective inhibitor of SERCA ATPases, a family of Ca²⁺-pumping ATPases present in the endoplasmic reticulum of all mammalian cells. We have studied some of the molecular features of thapsigargin responsible for its inhibitory action towards these Ca²⁺ ATPases. A series of thapsigargin analogues were synthesised and their inhibitory potencies determined using the uptake of ⁴⁵Ca²⁺ in bovine cerebellar microsomes as a sensitive marker of Ca²⁺ ATPase activity. An attenuation of the inhibitory potency relative to the parent compound was found ranging from slight to over 3 orders of magnitude. The inhibitory activity showed a very strong configuration dependence, a major contribution from the ester groups at C3 and C10, and an apparently minor contribution from the lactone ring substituents. The data are consistent with thapsigargin fitting to a sterically discriminating cleft involving the hydrophobic transmembrane region of the ATPase, and is compatible with available kinetic evidence of thapsigargin-mediated inhibition.