Actin plays central roles in cell motility through formation of the actin cytoskeleton. Recently, the very intriguing possibility that actin also contributes to processes in the cell nucleus has been emerging. To dissect its dynamics and functions, several actin-disrupting drugs have been widely and effectively employed. Among them, latrunculin-A has proved particularly useful, supplanting the classical drug cytochalasin-D. One reason is that latrunculin-A appears to bind only to actin monomers impairing the nucleotide exchange, the mode being simpler than with cytochalasin. This property may be especially crucial when studying actin functions as a monomer, as suggested for nuclear actin. Very importantly, actin mutations that cause cells to become resistant to the effects of latrunculin-A have been identified in budding yeast. However, it remains controversial as to whether all of the various phenotypes observed with latrunculin in mammalian cells more complicated than yeast are truly the consequence of its specific actions against actin. Here, we show that the expression of R183A D184A mutant beta-actin specifically confers resistance to the effects of latrunculin-A on actin cytoskeleton formation and cell growth in HeLa cells. The established system provides a strong tool to address the various functions of actin in mammalian cells.