Ca² plays an important role in the regulation of several important activities in different trypanosomatids. These parasites possess a Ca² transport system in the endoplasmic reticulum (ER) involved in Ca² homeostasis, which has been reported to be insensitive to thapsigargin, a classical inhibitor of the sarcoplasmic–ER Ca² adenosine triphosphatase (ATPase) (SERCA) in most eukaryotic cells. However, currently there is a controversy regarding the existence of a thapsigargin-sensitive ER Ca² store in these parasites. Therefore, we decided to explore the effect of this inhibitor using different methodological approaches. First, we selected Trypanosoma evansi as a parasite model to warrant the homogeneity of the population because this parasite has only a single life cycle, i.e., bloodstream-form trypomastigotes. Second, we compared the thapsigargin effect on Ca² homeostasis by spectrophotometrical Ca² measurements using 3 different approaches: whole-cell populations, cells that have been permeabilized by treatment with digitonin, and intact single cells. Our results demonstrate that a low concentration of thapsigargin induces Ca² release from intracellular Ca² stores in this parasite, which can be observed independently of the method used. Furthermore, the addition of thapsigargin before or after nigericin did not abolish its effect, showing that thapsigargin acts specifically on the ER. In conclusion, our results indicate the presence of a nonmitochondrial thapsigargin-sensitive Ca² store in T. evansi.