We examined the in vivo synthesis of brain phosphatidylcholine (PC) by the methylation of phosphatidylethanolamine (PE). [3H-methyl]methionine was infused i.c.v., by indwelling cannula, and brain samples were taken 0.5-18 h thereafter and assayed for [3H]PC, as well as for its biosynthetic intermediates [3H]phosphatidylmonomethylethanolamine ([3H]PMME) and [3H]phosphatidyldimethylethanolamine ([3H]PDME), and for [3H]lysophosphatidylcholine ([3H]LPC) and S-[3H]adenosylmethionine ([3H]SAM). Most of the [3H]PC (79-94%) was present ipsilateral to the infusion site, indicating that the radioactivity in the [3H]PC was primarily of intracerebral origin, and not taken up from the blood. Moreover, only very low levels of [3H]PC were attained in brains of animals receiving [3H]methionine i.p. and these levels were symmetrically distributed. [3H]PMME and [3H]PDME turned over with apparent half-lives of 2.2 h and 2.4 h. In contrast, the accumulation of brain [3H]PC was biphasic, suggesting the existence of two pools, the more labile of which turned over rapidly (t1/2 = 5 h) and was formed for as long as [3H]PMME and [3H]PDME are present in the brain, and another, which was distinguishable only at 18 h after the [3H]methionine infusion. (The latter pool may have been synthesized from [3H]choline that was released via the hydrolysis of some of the brain [3H]PC previously formed by the methylation of PE.) Subcellular fractionation of brain tissue obtained after in vivo labelling with [3H]methionine revealed that mitochondrial PC had the highest specific radioactivity (dpm per mumol total lipid phosphorus), and myelin the least. These observations affirm that rat brain does synthesize PC in vivo by methylating PE, and the technique provides an experimental system which may be useful for examining the physiological regulation of this process.