The Arabidopsis thaliana (L.) Heynh. ROOT HAIR DEFECTIVE3 (RHD3) gene has previously been shown to be essential for normal cell expansion [H. Wang et al. (1997) Genes Dev 11:799-811]. In this report, we demonstrated that mutation of the RHD3 gene in the Arabidopsis fragile fiber 4 (fra4) mutant caused a dramatic reduction in the wall thickness of fibers, vessels, and pith cells in the inflorescence stems and, concomitantly, a decrease in the mechanical strength of stems. The reduced wall thickness in the fra4 mutant was accompanied by an alteration in cell wall composition. Consistent with the defective fiber and vessel wall phenotypes, the RHD3 gene exhibited a strong expression in developing fiber and xylem cells. We showed that the Arabidopsis genome contains two additional RHD3-like genes, one of which was expressed specifically in flowers. In addition, we found that mutation of the RHD3 gene caused an alteration in the organization of the actin cytoskeleton but no effects on cortical microtubules. Our findings suggest an essential role of RHD3 in cell wall biosynthesis and actin organization, both of which are known to be important for cell expansion.