Eight functional actin genes are present in Arabidopsis: The functional characterization of these genes in loss-of-function mutants is difficult, because highly conserved isovariants are generally expressed in the same tissue. We isolated a novel semi-dominant mutant allele (act2-2D) of an actin gene, ACT2, with a missense mutation which causes an amino acid substitution at the surface of the ACT2 protein. ACT2 promoter::ACT2-2D transgenic plants showed the same phenotype as act2-2D, indicating that act2-2D is a dominant-negative mutant. act2-2D exhibited defects in the initiation and elongation of root hairs, the elongation of root epidermal cells, and growth in aerial portions. Specifically, radial cell expansion was reduced and occasional cell death occurred in trichoblasts but not in atrichoblasts of the root epidermis. In contrast, cell division patterns in the root meristem were not affected. act2-3, a loss-of-function ACT2 mutant, did not develop most of these morphological abnormalities. Actin filament (F-actin) bundles in root epidermal cells of act2-2D were shorter than in the wild type and in the loss-of-function mutant. We conclude that defective F-actin polymerization caused the aberrant cell morphology in a dominant-negative manner, and that ACT2 functions in cell elongation and root hair formation.