Synthetic cannabinoids are marketed as legal alternatives to Δ9-THC, and are a growing worldwide concern as these drugs are associated with severe adverse effects. Unfortunately, insufficient information regarding the physiological and pharmacological effects of emerging synthetic cannabinoids (ESCs) makes their regulation by government authorities difficult. One strategy used to evade regulation is to distribute isomers of regulated synthetic cannabinoids. This study characterized the pharmacological properties of a panel of ESCs in comparison to Δ9-THC, as well as six JWH-122 isomers relative to its parent compound (JWH-122-4). Two cell-based assays were used to determine the potency and efficacy of ESCs and a panel of reference cannabinoids. HEK293T cells were transfected with human cannabinoid receptor 1 (CB1) and pGloSensor-22F, and the inhibition of forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels was monitored in live cells. All ESCs examined were classified as agonists, with the following rank order of potency: Win 55,212-2 > CP 55,940 > JWH-122-4 > Δ9-THC ≈ RCS-4 ≈ THJ-2201 > JWH-122-5 > JWH-122-7 > JWH-122-2 ≈ AB-CHMINACA > JWH-122-8 > JWH-122-6 > JWH-122-3. Evaluation of ESC-stimulated Ca2+ transients in cultured rat primary hippocampal neurons confirmed the efficacy of four of the most potent ESCs (JWH-122-4, JWH-122-5, JWH-122-7 and AB-CHMINACA). This work helps regulatory agencies make informed decisions concerning these poorly characterized recreational drugs.