N-Acylethanolamines (NAEs) include palmitoylethanolamide, oleoylethanolamide and anandamide, which show anti-inflammatory/analgesic, anorexic and cannabimimetic actions, respectively. These lipid mediators are biosynthesized from N-acyl-phosphatidylethanolamine (NAPE) by NAPE-hydrolyzing phospholipase D (NAPE-PLD) as well as through multi-step pathways via lysoNAPE. We previously showed that genetic deletion of NAPE-PLD markedly increased NAPE levels and significantly decreased NAE levels in brain. Here, we aimed to examine the mechanisms for NAE biosynthesis in peripheral tissues. As compared to wild-type mice, NAPE-PLD–/– mice exhibited increased NAPE levels in heart, kidney and liver, but not in jejunum, suggesting a major role of NAPE-PLD in NAPE degradation in the three tissues. However, the deletion did not affect NAE levels in all the four tissues, showing compensation by other pathway(s). Accordingly, the tissue homogenates from NAPE-PLD–/– mice generated [14C]NAE and [14C]lysoNAPE from [14C]NAPE. These results suggested that the contribution of NAPE-PLD to NAE biosynthesis varies among tissues and might imply the possible presence of the tissue-specific mechanisms.