The objective of this investigation was to determine the distribution of cholinergic (acetyl‐cholinesterase, AChE) and noncholinergic markers in slow‐, fast‐, and mixed‐fiber containing muscles (soleus, SOL; extensor digitorum longus, EDL; and diaphragm, DIA, respectively). Noncholinergic markers included high‐energy phosphates (adenosine triphosphate, ATP; phosphocreatine, PCr; and their metabolites), and the activity of creatine kinase (CK) and lactate dehydrogenase (LDH) and their isoenzymes and subforms. All three types of muscles had only one CK isoenzyme, CK‐MM, which totally consisted of MM3 subform. Levels of these determinants were highest in EDL followed by DIA and least in SOL. Another objective was to determine alterations of these markers under the influence of acute carbofuran (1.5 mg/kg) or methyl parathion (MPTH, 5 mg/kg) toxicity. Rats receiving either insecticide showed cholinergic signs with maximal severity including muscle fasciculations and convulsions within 15–30 min that lasted for about 2 h. At 1 h postinsecticide injection, when AChE was maximally inhibited (81–96%), significant depletion of ATP and PCr was evident in muscles (DIA > SOL > EDL), and activities of CK‐MM and LDH were elevated in muscles and consequently in serum. Serum CK‐MM3 activity was markedly reduced with sequential increase in MM2 and MM1 subforms, probably due to induced higher carboxypeptidase activity. These findings suggested that (1) the differences in levels of biochemical constituents in muscles depend upon the fiber type, (2) anticholinesterase insecticide‐induced increased muscle activity produces characteristic changes in CK and LDH isoenzymes patterns, and (3) leakage of these enzymeslisoenzymes into serum is due to depletion of ATP and PCr, which are required to maintain the cell membrane permeability.