Recent evidence implicates anandamide as the endogenous ligand for the cannabinoid receptor. One purpose of this study was to determine the structural requirements for anandamide's receptor interaction and the influence of phenylmethylsulfonyl fluoride (PMSF), an enzyme inhibitor, on receptor affinity. A second objective was evaluation of the correlation between affinities of the analogs and in vivo pharmacological activities. The ability of anandamide and analogs to displace [3H]CP-55,940 ((-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3- hydroxylpropyl]cyclohexan-1-ol) was determined by a filtration assay. Displacement curves for anandamide in the presence of PMSF produced a Ki of 89 +/- 10 nM; without PMSF the Ki increased to 5400 +/- 1600 nM. Anandamide analogs were evaluated for their ability to produce antinociception and hypomotility. The levels of saturation of the anandamide structure were critical to receptor affinity and in vivo potency, with complete saturation and hydroxyl substitution with a fluorine moiety resulting in a compound with increased potency in the spontaneous activity and antinociception assays. Substitution of the hydroxyl with a fluorine atom increased affinity only in the presence of PMSF and reduced potency in the antinociception assay. Ethanolamide substitution with bromobenzenesulfonamide produced an inactive compound in all assays. Increasing the length of the N-substituent by one or two carbons decreased receptor binding affinity and potency in the tail-flick assay only. Certain structural modifications, such as methylations, allowed the analogs to retain affinity without the addition of PMSF. Linear correlation between the behavioral and binding assays were performed, and the greatest correlation was obtained with compounds that were either very potent or inactive.