ABSTRACT: Rho/Ras family small GTPases are known to regulate numerous cellular processes, including cytoskeletal reorganization, cell proliferation, and cell differentiation. These processes are also controlled by Ca2+, and consequently, cross talk between these signals is considered likely. However, systematic quantitative evaluation has not yet been reported. To fill this gap, we constructed optogenetic tools to control the activity of small GTPases (RhoA, Rac1, Cdc42, Ras, Rap, and Ral) using an improved light-inducible dimer system (iLID). We characterized these optogenetic tools with genetically encoded red fluorescence intensity-based small GTPase biosensors and confirmed these optogenetic tools' specificities. Using these optogenetic tools, we investigated calcium mobilization immediately after small GTPase activation. Unexpectedly, we found that a transient intracellular calcium elevation was specifically induced by RhoA activation in RPE1 and HeLa cells. RhoA activation also induced transient intracellular calcium elevation in MDCK and HEK293T cells, suggesting that generally RhoA induces calcium signaling. Interestingly, the molecular mechanisms linking RhoA activation to calcium increases were shown to be different among the different cell types: In RPE1 and HeLa cells, RhoA activated phospholipase C epsilon (PLCε) at the plasma membrane, which in turn induced Ca2+ release from the endoplasmic reticulum (ER). The RhoA-PLCε axis induced calcium-dependent nuclear factor of activated T cells nuclear translocation, suggesting that it does activate intracellular calcium signaling. Conversely, in MDCK and HEK293T cells, RhoA-ROCK-myosin II axis induced the calcium transients. These data suggest universal coordination of RhoA and calcium signaling in cellular processes, such as cellular contraction and gene expression.