Aims: To determine the effect of articaine on sarcoendoplasmic reticulum calcium adenosine triphosphatase (SERCA) isoforms of the medial pterygoid muscle. Methods: Native SERCA from the medial pterygoid muscles of 24 rabbits was isolated by ultracentrifugation, and its isoforms were purified by chromatography and assessed by enzyme-linked immunosorbent assay (ELISA). SERCA activity and calcium transport capability were determined by using colorimetric and radioisotopic methods. The mean ± standard deviation (SD) half maximal inhibitory concentration (IC50) of articaine was determined for each isoform, and these values were compared by using analysis of variance (ANOVA) (P .05). Results: The native SERCA preparation consisted of 34% SERCA1a, 53% SERCA2a, 10% SERCA2b, and 3% combined SERCA3 and SERCA1b. Articaine caused inhibition of activity and calcium uptake in the native SERCA preparation and in each of the purified isoforms. The IC50 (mM) values for enzymatic activity were: SERCA1a 22.0 ± 2.3 > SERCA2a 16.4 ± 2.4 > SERCA2b 11.3 ± 1.9, and 15.1 ± 2.1 for native SERCA. For calcium transport, IC50 values were: SERCA1a 31.1 ± 3.3 > SERCA2a 24.8 ± 1.8 > SERCA2b 21.5 ± 1.5, and 25.2 ± 3.2 for native SERCA. IC50 values for inhibition of enzymatic activity were significantly different among the purified isoforms, but only the value obtained for SERCA1a was significantly different compared to native SERCA. For inhibition of calcium transport, IC50 values for both SERCA2a and SERCA2b differed significantly compared to SERCA1a, and the value for SERCA1a was significantly different compared to native SERCA. The most articaine-sensitive isoform was SERCA2b, and the native preparation showed sensitivity similar to SERCA2a. Conclusion: The differential inhibition of articaine on medial pterygoid SERCA isoforms is evident at concentrations lower than used in current dental practice (125 mM) and accounts for anesthetic myotoxicity. Muscle relaxation likely becomes impaired as a result of increased calcium levels in the myoplasm due to the decreased activity and calcium transport caused by the inhibition of SERCA. Keywords: articaine, local anesthetics, masticatory muscles, medial pterygoid muscle, SERCA