Previous studies have demonstrated that addition of glutathione at the penultimate Cys374 residue of actin results in filaments with diminished mechanical stability. In the present work substitutions introducing a negatively charged (Asp and Glu) or a neutral (Ala) amino acid at position 374 of the human beta-actin and tagged at the N-terminus with the flag epitope were studied by transient transfections into Ishikawa human endometrial and opossum kidney cells. Immunofluorescence revealed that microfilaments which incorporated negatively charged mutants were partially to severely disorganized when compared to the almost well-formed actin-Ala374 filaments or the wild type actin filaments. Furthermore, microfilaments containing either negatively charged mutant were more sensitive to the destabilizing action of cytochalasin B. In addition, Triton fractionation resealed a considerable reduction of flag-actin content in the Triton insoluble fraction for cells expressing Asp374 or Glu374 mutant compared to wild type actin. These results demonstrate that negatively charged amino acid residues at the exposed C-terminal tail strongly affect actin microfilament organization and dynamics in vivo.