Mitochondrial cardiolipin synthase catalyzes the transfer of a phosphatidyl moiety from phosphatidyl-CMP (PtdCMP) to phosphatidylglycerol (PtdGro) in the presence of specific divalent cations. The synthase was solubilized from Saccharomyces cerevisiae mitochondria and purified about 300-fold. The partially enzyme was part of a medium-size, mixed micelle which had to bind to a foreign substrate/detergent micelle before catalysis could occur. The kinetics of cardiolipin synthase were studied by changing the molar fraction of substrate in the micelles. The enzyme obeyed Michaelis-Menten kinetics in relation to PtdCMP with a Km of 0.03 mol%. PtdGro caused sigmoidal kinetics with a low apparent affinity. It is speculated that it was involved in docking the enzyme to the substrate/detergent micelle. Cardiolipin synthase did not catalyze isotope exchange between [14C]CMP and PtdCMP, virtually excluding a ping-pong catalytic mechanism. Mg2+ stimulated the activity by increasing the turnover number rather than the substrate affinity, a mechanism which was also found for the Co(2+)-activation of rat liver cardiolipin synthase. It is concluded that a direct association of the metal ion and the enzyme forms the active cardiolipin synthase which has a very high affinity for PtdCMP and a lower affinity for PtdGro.