In response to various environmental stress conditions, plants rapidly form the intracellular lipid second messenger phosphatidic acid (PA). It can be generated by two independent signalling pathways via phospholipase D (PLD) and via phospholipase C (PLC) in combination with diacylglycerol kinase (DGK). In the green alga Chlamydomonas, the phospholipid substrates for these pathways are characterized by specific fatty acid compositions. This allowed us to establish: (i) PLD's in vivo substrate preference; and (ii) PLD's contribution to PA formation during stress signalling. Accordingly, G-protein activation (1 micro m mastoparan), hyperosmotic stress (150 mm NaCl) and membrane depolarization (50 mm KCl) were used to stimulate PLD, as monitored by the accumulation in 5 min of its unique transphosphatidylation product phosphatidylbutanol (PBut). In each case, PBut's fatty acid composition specifically matched that of phosphatidylethanolamine (PE), identifying this lipid as PLD's favoured substrate. This conclusion was substantiated by analysing the molecular species by electrospray ionization-mass spectrometry (ESI-MS/MS), which revealed that PE and NaCl-induced PBut share a unique (18 : 1)2-structure. The fatty acid composition of PA was much more complex, reflecting the different contributions from the PLC/DGK and PLD pathways. During KCl-induced stress, the PA rise was largely accounted for by PLD activity. In contrast, PLD's contribution to hyperosmotic stress-induced PA was less, being approximately 63% of the total increase. This was because the PLC/DGK pathway was activated as well, resulting in phosphoinositide-specific fatty acids and molecular species in PA.