Serine decarboxylation as a source of glycerophospholipid ethanolamine is known to occur in mammals. However, early investigators failed to demonstrate the pathway in brain. In the present study serine is shown to be decarboxylated to glycerophospholipid ethanolamine in myelinating organ cultures of rat cerebellum up to 32 days in vitro. The pattern of incorporation of L-[3-14C]serine into culture phospholipids strongly suggests a precursor-product relationship between serine glycerophospholipids (SGP) and ethanolamine glycerophospholipids (EGP), with serine label appearing in the ethanolamine moiety of EGP. The time course of labelling was similar for both acid-stable and acid-labile EGP. In contrast DL-[1-14C]serine failed to label EGP significantly due to the loss of serine carbon C1 on decarboxylation. Through the systematic hydrolysis of phospholipids from cerebellar cultures incubated with L-[3-14C], it was clear that in SGP, acid-stable EGP, and acid-labile EGP greater than to 70% of radiolabel resides in the base moiety of each of these molecular species. It is proposed that serine decarboxylation as a source of EGP ethanolamine may be important in the early stages of brain development.