TGF signaling is involved in the development and regulation of multiple organ systems and cellular signaling pathways. We recently demonstrated that TGF regulates the response of atrial myocytes to parasympathetic stimulation. Here, TGF₁ is shown to inhibit expression of the M₂ muscarinic receptor (M₂) which plays a critical role in the parasympathetic response of the heart. This effect is mimicked by overexpression of a dominant negative mutant of RhoA and by the RhoA kinase inhibitor Y27632 while adenoviral expression of a DA-RhoA reverses TGF inhibition of M₂ expression. TGF₁ also mediates a decrease in GTP-bound RhoA and a reciprocal increase in the expression of the RhoA GTPase activating protein, p190RhoGAP, while total RhoA is unchanged. Inhibition of M₂ promoter activity by TGF₁ is mimicked by overexpression of p190RhoGAP while a dominant negative mutant of p190RhoGAP reverses this effect of TGF₁. In contrast to atrial myocytes, in mink lung epithelial cells, in which TGF signaling through activation of RhoA has been previously identified, TGF₁ stimulated an increase in GTP-bound RhoA in association with a reciprocal decrease in the expression of p190RhoGAP. Both effects demonstrated a similar dose dependence on TGF₁. Thus TGF regulation of M₂ muscarinic receptor expression is dependent on RhoA and TGF regulation of p190RhoGAP expression may be a cell type specific mechanism for TGF signaling through RhoA.