The established pathways from serine to ethanolamine are indirect and involve decarboxylation of phosphatidylserine. Here we show that plants can decarboxylate serine directly. Using a radioassay based on ethanolamine (Etn) formation, pyridoxal 5'-phosphate-dependent l-serine decarboxylase (SDC) activity was readily detected in soluble extracts from leaves of diverse species, including spinach, Arabidopsis, and rapeseed. A putative Arabidopsis SDC cDNA was identified by searching GenBank for sequences homologous to other amino acid decarboxylases and shown by expression in Escherichia coli to encode a soluble protein with SDC activity. This cDNA was further authenticated by complementing the Etn requirement of a yeast psd1 psd2 mutant. In a parallel approach, a cDNA was isolated from a rapeseed library by its ability to complement the Etn requirement of a yeast cho1 mutant and shown by expression in E. coli to specify SDC. The deduced Arabidopsis and rapeseed SDC polypeptides are 90% identical, lack obvious targeting signals, and belong to amino acid decarboxylase group II. Recombinant Arabidopsis SDC was shown to exist as a tetramer and to contain pyridoxal 5'-phosphate. It does not attack d-serine, l-phosphoserine, other l-amino acids, or phosphatidylserine and is not inhibited by Etn, choline, or their phosphoesters. As a soluble, pyridoxal 5'-phosphate enzyme, SDC contrasts sharply with phosphatidylserine decarboxylases, which are membrane proteins that have a pyruvoyl cofactor.