The apical membrane is an important site of mercury toxicity in shark rectal gland tubular cells. We compared the effects of mercury and other thiol-reacting agents on shark CFTR (sCFTR) and human CFTR (hCFTR) chloride channels using two-electrode voltage clamping of cRNA microinjected Xenopus laevis oocytes. Chloride conductance was stimulated by perifusing with 10µM forskolin (FOR) and 1mM IBMX and then thio-reactive species were added. In oocytes expressing sCFTR, FOR + IBMX mean stimulated Cl^--conductance was inhibited 69% by 1µM mercuric chloride, and 78% by 5µM mercuric chloride (IC₅₀ of 0.8 µM). Despite comparable stimulation of conductance, human CFTR was insensitive to 1µM HgCl₂ and maximum inhibition was 15% at the highest concentration used (5µM). Subsequent exposure to glutathione (GSH) did not reverse the inhibition of sCFTR by mercury, but dithiothreitol (DTT) completely reversed this inhibition. Zinc (50-200µM) also reversibly inhibited sCFTR (40-75%) but did not significantly inhibit hCFTR. Similar inhibition of sCFTR but not hCFTR was observed with an organic mercurial, p-chloromercuriphenylsulfonic acid (pCMBS). The first membrane spanning domain (MSD1) of sCFTR contains two unique cysteines, C102 and C303. A chimeric construct replacing MSD1 of hCFTR with the corresponding sequence of sCFTR was highly sensitive to mercury. Site specific mutations introducing the first but not the second shark unique cysteine into hCFTR MSD1 resulted in full sensitivity to mercury. These experiments demonstrate a profound difference in the sensitivity of shark versus human CFTR to inhibition by three thiol-reactive substances, an effect that involves C102 in the shark orthologue.