The endothelial ET_B receptor subtype is critical for renal vasodilation induced by relaxin in nonpregnant rats and during pregnancy (the latter via endogenous circulating relaxin). Here, we tested whether expression of vascular endothelin B (ET_B) receptor protein is regulated by relaxin. Small renal arteries were harvested from virgin and midterm pregnant rats, as well as from nonpregnant rats that were administered recombinant human relaxin (rhRLX) at 4 µg/h or vehicle for 5 days or for 4-6 h. Small renal arteries dissected from additional virgin rats were incubated in vitro with rhRLX or vehicle for 3 h at 37°C. ET_B expression was also evaluated in cultured human endothelial cells: aortic (HAEC), coronary (HCAEC), umbilical vein (HUVEC), and dermal microvascular endothelial cells (HMVEC). Cells were incubated for 4, 8, or 24 h with rhRLX (5, 1, or 0.1ng/ml) or with vehicle. ET_B protein expression in arteries and cells was evaluated by Western analysis. No regulation of ET_B expression was observed in small renal arteries in any of the experimental protocols. Nor was there an increase in the vasorelaxation response to ET-3 in small renal arteries incubated in vitro with rhRLX. rhRLX only sporadically altered ET_B expression in HCAEC and HUVEC at certain time points or doses, and no regulation was observed in HAEC or HMVEC. These results suggest that regulation of ET_B receptor protein has little or no role in relaxin stimulation of the endothelial ET_B/NO vasodilatory pathway.