We have identified a microsomal phospholipid as N-acylphosphatidylethanolamine (NAPE) that was radiolabeled following incubation of 1-day-old cotyledons of cotton seedlings with [14C]ethanolamine. Radiolabeled NAPE comigrated with commercially available L-alpha-dipalmitoyl phosphatidyl(N-palmitoyl)ethanolamine (std-NAPE) in one- and two-dimensional TLC. This NAPE comprised 7.2 +/- 1.0% (by weight) of microsomal phospholipids when hot isopropanol was used to inactivate endogenous phospholipases prior to extraction of lipids. In vitro degradation of putative cottonseed radiolabeled NAPE by Streptomyces chromofuscus phospholipase D resulted in production of a ninhydrin-reactive, radiolabeled lipid which comigrated with N-acylethanolamine (NAE) that was produced from a similar enzymatic cleavage of std-NAPE. Transmethylation of cottonseed radiolabeled NAE yielded radiolabeled ethanolamine and fatty acid methyl esters, nearly all of which were saturated (myristate, palmitate, and stearate together were 92% of the acyl components of cottonseed NAE). Positional analysis and relative abundance of the O-acyl groups of cottonseed microsomal NAPE were determined following a double enzymatic cleavage with Trimeresurus flavoviridis venom (phospholipase A2 activity) and S. chromofuscus phospholipase D. We substantiated our identification of cottonseed NAPE by 1H NMR spectroscopy and by mass spectrometry (fast-atom-bombardment ionization and tandem MS, FAB-MS/MS). Radiolabeled NAPE was synthesized in vivo in varying amounts from [14C]ethanolamine applied to maturing seeds of cotton and soybean, cotyledons of dark-grown cotton and soybean seedlings, cotyledons of light-grown okra, cotton and soybean seedlings, endosperm tissue of castor bean, and suspension cell cultures of rice. In pulse-chase radiolabeling experiments in cotyledons of 1-day-old cotton seedlings, radiolabeled NAPE increased and radiolabeled phosphatidylethanolamine (PE) decreased over a 12-h chase period (in the dark or light), suggesting that NAPE was synthesized from PE in vivo. In vitro, the synthesis of NAPE from PE (radiolabeled in vivo) proceeded in a linear fashion in microsomes of cotton cotyledons with or without 1 mM EGTA and with or without 1 mM CaCl2 for 90 min. NAPE was synthesized in vitro from PE synthesized by the exchange pathway (microsomes preincubated with [14C]++e+thanolamine) and from PE synthesized by the nucleotide pathway (microsomes preincubated with [14C]CDPethanolamine). Collectively, our data indicate that (a) NAPE is a widespread, natural phospholipid component of plants, (b) NAPE is synthesized in vivo under normal physiological growth conditions in cotyledons of cotton seedlings, (c) NAPE is localized and synthesized in cottonseed microsomes, and (d) NAPE is likely synthesized by a direct acylation of PE.