The amphibian urea transporter (fUT) shares many properties with the mammalian urea transporters (UT) derived from UT-A and UT-B genes. The transport of urea by fUT is inhibited by the mercurial agent p-chloromercuribenzenesulfonic acid (pCMBS). We found that in oocytes expressing cRNA encoding fUT, a five minute pre-incubation in 0.5mM mercury chloride (HgCl₂) also significantly reduced urea uptake. The transport of urea by fUT was rendered mercury (Hg⁺⁺) insensitive by mutating either of the residues C185 or H187, both of which lie within the M-I region (close to the hypothetical UT pore). In oocytes expressing a mixture of the C185 and H187 mutants, Hg⁺⁺-sensitivity was re-established. The transport of urea by the mouse UTs, mUT-A2 and mUT-A3 was not sensitive to Hg⁺⁺. Introducing cysteine residues analogous to that mutated in fUT into mUT-A2 or mUT-A3 did not induce Hg⁺⁺ sensitivity. Additionally introducing the double cysteine, histidine mutations into mUT-A2 or mUT-A3 still did not induce Hg⁺⁺ sensitivity, indicating that a region outside of the M-I region also contributes to the Hg⁺⁺ induced block of fUT. Using a series of chimeras formed between UT-A3 and fUT we found that as well as C185 and H187, residues within the C-terminal of fUT determine Hg⁺⁺ sensitivity and we propose that differences in the folding of this region between fUT and mUT-A2/mUT-A3 allow access of Hg⁺⁺ to the fUT channel pore.