Cardiolipin, a unique dimeric phospholipid of bacteria and mitochondria, can be synthesized by two alternative pathways discovered in rat and Escherichia coli, respectively. In mitochondrial preparations from fungi (Saccharomyces cerevisiae, Neurospora crassa), higher plants (Phaseolus aureus), molluscs (Mytilus edulis) and mammals (rat liver, bovine adrenal gland), cardiolipin was synthesized from CDP-diacylglycerol and phosphatidylglycerol, suggesting a common eukaryotic mechanism of cardiolipin formation which is in contrast to the prokaryotic biosynthesis from two molecules of phosphatidylglycerol. All mitochondrial cardiolipin synthases were inhibited by lysophosphatidylglycerol, were insensitive to N-ethylmaleimide and required divalent cations, although they had different cation specificities. The molecular species of cardiolipin from rat liver, bovine heart, S. cerevisiae and N. crassa were analysed by high-performance liquid chromatography of the derivative 1,3-bis[3'-sn-phosphatidyl]-2-benzoyl-sn-glycerol dimethyl ester. Cardiolipins from these organisms contained mainly monounsaturated or diunsaturated chains with 16 or 18 carbon atoms, resulting in a relatively homogeneous distribution of double bonds and carbon numbers among the four acyl positions. About half of the molecular species were symmetrical, i.e. they combined two identical diacylglycerol moieties. In N. crassa, the same species pattern was found at growth temperatures of 25 degrees C and 37 degrees C. Tentative molecular models were created for the most abundant molecular species and subjected to energy minimization. Geometric data, derived from these models, suggested similarities in the gross structure of the major cardiolipin species from different sources.