Small GTPases of the Rho subfamily have been implicated in many physiological and pathological processes in various cell types including embryonic stem cells (ESCs). In the present study we performed a functional analysis of the promoters of the RhoA and the RhoB genes in order to identify regulatory elements that are important for their transcriptional control in ESCs. We show that RhoA mRNA levels were significantly higher compared with the RhoB mRNA levels in ESCs as well in various cancer cell lines and this difference could be accounted for by differences in the activities of the corresponding promoters. Deletion analysis of the RhoA and RhoB promoters in ESCs revealed that the proximal regions contain regulatory elements that are critical for their activity. Both proximal promoters contain CCAAT boxes and mutagenesis of these elements decreased significantly the activity of both promoters suggesting a coordinated regulation of the two genes by CCAAT box binding factors. Finally, we show that both genes are subjects to autoregulation in ESCs and in the case of RhoB, this autoregulation requires the GTPase activity of the Rho proteins. Understanding the mechanisms that control the transcription of Rho GTPases in ESCs may shed new light into the still unknown roles of these proteins in stem cell functions. (C) 2017 Elsevier Inc. All rights reserved.