We have investigated the biosynthesis of long-chain N-acylethanolamines (NAEs) from endogenous substrates in rat testes membranes with special emphasis on anandamide (20:4n-6 NAE), a cannabinoid receptor agonist. Incubation of various membrane preparations with 5 mM Ca2+ produced both N-acyl phosphatidylethanolamine (N-acyl PE) and NAE with primarily (approximately 85%) N-palmitoyl groups (16:0 NAE) and less than 2% 20:4n-6 NAE. In contrast, incubation of these membranes with 5 mM EGTA and 10 mM ethanolamine had little effect on N-acyl PE composition but yielded NAEs whose major constituent (32-37%) was anandamide. Incubations with [1,1,2,2,-2H4]ethanolamine in media containing 40% H2(18)O showed that the Ca(2+)-independent NAE synthesis occurred by direct condensation of ethanolamine with free fatty acids present in the membrane preparation. This biosynthetic activity occurred at ethanolamine concentrations as low as 50 microM and exhibited substrate selectivity for arachidonate which increased with increasing ethanolamine concentrations. The results of inhibitor experiments suggest that the Ca(2+)-independent NAE synthesis was catalyzed by the NAE amidohydrolase acting in reverse. This condensation reaction could be important in agonist-induced anandamide synthesis for cell signalling through cannabinoid receptors.