Key points Cytosolic, but not matrix, Mg2+ inhibits mitochondrial Ca2+ uptake through the Ca2+ uniporter (CU). The majority of mitochondrial Ca2+ uptake under physiological levels of cytosolic Ca2+ and Mg2+ is through a fast uptake pathway, namely the ryanodine receptor (RyR)‐type channel (RTC), that has characteristics similar to the ryanodine receptor. Modulation of mitochondrial RTC adaptation and opening probability by cytosolic Mg2+ is not robust, in contrast to that of cardiac sarcoplasmic reticulum RyR. Model analysis of the mitochondrial Ca2+ sequestration system further validates the existence of two different classes of Ca2+ buffering proteins, i.e. Class 1 and Class 2. The Ca2+ buffering capacity of Class 1 protein is auto‐regulated by the rate at which Ca2+ is taken up by cardiac mitochondria. The quantitative framework suggests differential roles for the two modes of Ca2+ uptake pathways: CU–Ca2+ buffering, and RTC–Ca2+ modulated bioenergetics. Abstract Cardiac mitochondria can act as a significant Ca2+ sink and shape cytosolic Ca2+ signals affecting various cellular processes, such as energy metabolism and excitation–contraction coupling. However, different mitochondrial Ca2+ uptake mechanisms are still not well understood. In this study, we analysed recently published Ca2+ uptake experiments performed on isolated guinea pig cardiac mitochondria using a computer model of mitochondrial bioenergetics and cation handling. The model analyses of the data suggest that the majority of mitochondrial Ca2+ uptake, at physiological levels of cytosolic Ca2+ and Mg2+, occurs through a fast Ca2+ uptake pathway, which is neither the Ca2+ uniporter nor the rapid mode of Ca2+ uptake. This fast Ca2+ uptake component was explained by including a biophysical model of the ryanodine receptor (RyR) in the computer model. However, the Mg2+‐dependent enhancement of the RyR adaptation was not evident in this RyR‐type channel, in contrast to that of cardiac sarcoplasmic reticulum RyR. The extended computer model is corroborated by simulating an independent experimental dataset, featuring mitochondrial Ca2+ uptake, egress and sequestration. The model analyses of the two datasets validate the existence of two classes of Ca2+ buffers that comprise the mitochondrial Ca2+ sequestration system. The modelling study further indicates that the Ca2+ buffers respond differentially depending on the source of Ca2+ uptake. In particular, it suggests that the Class 1 Ca2+ buffering capacity is auto‐regulated by the rate at which Ca2+ is taken up by mitochondria.