Phosphatidylserine decarboxylase (PSD) is an important enzyme in the synthesis of phosphatidylethanolamine in both prokaryotes and eukaryotes. The cloned bacterial gene encodes an integral membrane protein that is first made as a proenzyme, and subsequently proteolyzed to an alpha subunit, containing a pyruvoyl prosthetic group, and a beta subunit. Two types of decarboxylases are found in yeast, PSD1 and PSD2, that localize to the inner mitochondrial membrane and the Golgi/vacuole membrane, respectively. The mammalian enzyme is also found in the inner mitochondrial membrane. The yeast genes and mammalian cDNA have been cloned and sequenced. The yeast genes contain 5' sequences associated with regulation of expression by inositol and choline. The yeast PSD1 and the mammalian PSD both contain an LGST amino acid motif that identifies the site of proteolysis and pyruvoyl prosthetic group attachment in the bacterial enzyme. The yeast PSD1 and mammalian PSD also have mitochondrial targeting and inner membrane sorting sequences. Processing intermediates have been defined in the mammalian enzyme that correspond to the sequential removal of the mitochondrial targeting and inner membrane sorting sequence, followed by formation of the alpha and beta subunits. In contrast, the PSD2 enzyme contains a putative Golgi localization/retention sequence and a C2 homology domain, in addition to predicted alpha and beta subunits. The transport requirements for substrate access to the PSD enzymes have provided important information about lipid trafficking, and the availability of yeast mutants is likely to provide important new genetic selections in the future.