The toxicity of abamectin to the Iranian populations of two-spotted spider mite, Tetranychus urticae, collected from Isfahan (ISR) and Guilan (GUS2) provinces were assayed using the residual contact vial (RCV) bioassay. The results interestingly showed more than 12755-fold resistance to abamectin in the ISR population of T. urticae compared with the susceptible GUS2 population. The synergistic effects of Triphenyl Phosphate (TPP), Piperonyl Butoxide (PBO) and Diethyl Maleate (DEM) were carried out to determine the involvement of esterase, mixed functional oxidase (MFO) and glutathione S-transferase (GST) in resistance mechanisms, respectively. Due to very high levels of resistance in the ISR population, it was not possible to calculate LC50 value nor to accurately assess the effects of synergists on this population. When alpha-naphthyl acetate (alpha-NA) was used as a substrate, the difference in esterase activity between the ISR and GUS2 populations was statistically significant, but low. Kinetic studies also indicated that the alpha-NA hydrolyzing esterases in the ISR population were different from those of the GUS2 population. Activity of GST in the ISR population was 2-fold more than that in the GUS2 population, and Km and Vmax values of the ISR population to a 1-choloro-2,4-dinitrobenzene (CDNB) substrate were 1.44 and 1.21 times lower and higher than those of the susceptible counterpart, respectively. The amount of heme content in the ISR population was 1.26 times more than that in the GUS2 population. Finally, comparing the nucleotide sequences of one of the transmembrane regions of the glutamate-gated chloride channel (GluCl1) from the two studied populations showed no substitutions in the deduced amino acid sequence of this region in the ISR population.