N-Acyl ethanolamines (NAEs) constitute a large and diverse class of signaling lipids that includes the endogenous cannabinoid anandamide. Like other lipid transmitters, NAEs are thought to be biosynthesized and degraded on-demand rather than being stored in vesicles prior to signaling. The identification of enzymes involved in NAE metabolism is therefore imperative to achieve a complete understanding of this lipid signaling system and control it for potential therapeutic gain. Recently, an N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) was identified as a candidate enzyme involved in the biosynthesis of NAEs. Here, we describe the generation and characterization of mice with a targeted disruption in the NAPE-PLD gene [NAPE-PLD(-/-) mice]. Brain tissue from NAPE-PLD(-/-) mice showed more than a 5-fold reduction in the calcium-dependent conversion of NAPEs to NAEs bearing both saturated and polyunsaturated N-acyl chains. However, only the former group of NAEs was decreased in level in NAPE-PLD(-/-) brains, and these reductions were most dramatic for NAEs bearing very long acyl chains (>or=C20). Further studies identified a calcium-independent PLD activity in brains from NAPE-PLD(-/-) mice that accepted multiple NAPEs as substrates, including the anandamide precursor C20:4 NAPE. The illumination of distinct enzymatic pathways for the biosynthesis of long chain saturated and polyunsaturated NAEs suggests a strategy to control the activity of specific subsets of these lipids without globally affecting the function of the NAE family as a whole.