Primary fatty acid amides (PFAMs) and N-acylglycines (NAGs) are important signaling molecules in the mammalian nervous system, binding to many drug receptors and demonstrating control over sleep, locomotor activity, angiogenesis, vasodilatation, gap junction communication, and many other processes. Oleamide is the best-studied of the PFAMs, while the in vivo activity of the others is largely unstudied. Even less is known about the NAGs, as their discovery as novel compounds is much more recent due to low endogenous levels. Herein is described extraction and quantification techniques for PFAMs and NAGs in cultured cells and media using solvent extraction combined with solid phase extraction (PFAM) or thin layer chromatography (NAG), followed by gas chromatography-mass spectroscopy to isolate and quantify these lipid metabolites. The assays were used to examine the endogenous amounts of a panel of PFAMs as well as the conversion of corresponding free fatty acids (FFAs) to PFAMs over time in several cell lines. The cell lines demonstrated the ability to convert all FFAs, including a non-natural FFA, and an ethanolamine to the corresponding PFAM. Different patterns of relative amounts of endogenous and FFA-derived PFAMs were observed in the cell lines tested. Essential to identifying therapeutic targets for the many disorders associated with PFAM signaling is understanding the mechanism(s) of PFAM and NAG biosynthesis. Enzyme expression studies were conducted to determine potential metabolic enzymes in the model cell lines in an attempt to understand the mechanism(s) of PFAM biosynthesis. It was found that two of the cell lines which show distinct metabolisms of PFAMs also demonstrate unique enzyme expression patterns, and candidate enzymes proposed to perform PFAM and NAG metabolism are described. RNAi knockdown studies revealed further information about the metabolism of PFAMs and calls into question the recently proposed involvement of cytochrome c. Isotopic labeling studies showed there are two pathways for PFAM formation. A novel enzyme is likely to be involved in formation of NAGs from acyl-CoA intermediates.