Mitochondrial Ca²⁺ sequestration is a well-known process that is involved in various physiological and pathological mechanisms. Using isolated suspended mitochondria one unique mitochondrial Ca²⁺ uniporter was considered to account ubiquitously for the transfer of Ca²⁺ into these organelles. However, by applying alternative techniques for measuring mitochondrial Ca²⁺ uptake evidences for molecularly distinct mitochondrial Ca²⁺ carriers accumulated recently. Herein we compared different methodical approaches of studying mitochondrial Ca²⁺ uptake. Patch clamp technique on mitoplasts from endothelial and HeLa cells revealed the existence of three and two mitoplast Ca²⁺ currents (I_CaMito), respectively. According to their conductance, these channels were named small (s-), intermediate (i-), large (l-) and extra-large (xl-) mitoplast Ca²⁺ currents (MCC). i-MCC was found in mitoplasts of both cell types whereas s-MCC and l-MCC or xl-MCC were/was exclusively found in mitoplasts from endothelial cells or HeLa cells. The comparison of mitochondrial Ca²⁺ signals, measured either indirectly by sensing extra-mitochondrial Ca²⁺ or directly by recording changes of the matrix Ca²⁺, showed different Ca²⁺ sensitivities of the distinct mitochondrial Ca²⁺ uptake routes. Subpopulations of mitochondria with different Ca²⁺ uptake capacities in intact endothelial cells could be identified using Rhod-2/AM. In contrast, cells expressing mitochondrial targeted pericam or cameleon (4mtD3cpv) showed homogeneous mitochondrial Ca²⁺ signals in response to cell stimulation. The comparison of different experimental approaches and protocols using isolated organelles, permeabilized and intact cells, pointed to cell-type specific and versatile pathways for mitochondrial Ca²⁺ uptake. Moreover, this work highlights the necessity of the utilization of multiple technical approaches to study the complexity of mitochondrial Ca²⁺ homeostasis.