Phosphatidic acid (PtdOH) is a key intermediate in glycerolipid biosynthesis. Two different pathways are known for de novo formation of this compound, namely (a) the Gro3P (glycerol 3-phosphate) pathway, and (b) the GrnP (dihydroxyacetone phosphate) pathway. Whereas the former route of PtdOH synthesis is present in bacteria and all types of eukaryotes, the GrnP pathway is restricted to yeast and mammalian cells. In this review article, we describe the enzymes catalyzing de novo formation of PtdOH, their properties and their occurrence in different cell types and organelles. Much attention has recently been paid to the subcellular localization of enzymes involved in the biosynthesis of PtdOH. In all eukaryotic cells, microsomes (ER) harbour the complete set of enzymes catalyzing these pathways and are thus the usual organelle for PtdOH formation. In contrast, the contribution of mitochondria to PtdOH synthesis is restricted to certain enzymes and depends on the cell type. In addition, chloroplasts of plants, lipid particles of the yeast, and peroxisomes of mammalian cells are significantly involved in PtdOH biosynthesis. Redundant systems of acyltransferases, the interplay of organelles, regulation of the pathway on the compartmental level, and finally the contribution of alternative pathways (phosphorylation of diacylglycerol and cleavage of phospholipids by phospholipases) to PtdOH biosynthesis appear to be required for the balanced formation of this important lipid intermediate. Dysfunction of enzymes involved in PtdOH synthesis can result in severe defects of various cellular processes. In this context, the possible physiological role(s) of PtdOH and its related metabolites, lysophosphatidic acid and diacylglycerol, will be discussed.