Objective:To investigate the effect of cellular ATP depletion on actin phosphorylation in rat renal proximal tubule cells(NRK52E). Methods:NRK52E cells were treated with 0.1 μmol/L Antimycin A to established a cellular ATP depletion model. The contents of G-actin(in cytosolic fraction)and F-actin(in cytoskeletal fraction)in cells were evaluated by Western blot. Two-dimensional electrophoresis was used to separate phosphorylated actin from unphosphorylated actin of cells. With immunoprecipitation(IP)and Western blot analysis,residues of actin phosphorylated was explored by using monoclonal anti-phosphoserine,anti-phosphotyrosine and anti-phosphothreonine antibody. Results:The cellular F-actin was increased and G-actin was decreased after ATP depletion. The proportion of phosphorylated actin after ATP depletion was higher than that of control cells. As compared to the control cells,the level of actin phosphorylation on thyrosine residue(s) was significantly increased during cellular ATP depletion,and these changes were paralleled with the severity of cellular ATP depletion. Conclusion:The thyrosine phosphorylation of actin of NRK52E cells may play an important role in regulation of polymerization of actin during ATP depletion.