Most vertebrates, including cartilaginous fishes, maintain their plasma SO₄^2- concentration ([SO₄^2-]) within a narrow range of 0.2-1 mM. As seawater has a [SO₄^2-] about 40 times higher than that of the plasma, SO₄^2- excretion is major role of the kidneys in marine teleost fishes. It has been considered that cartilaginous fishes also excrete excess SO₄^2- via the kidney. However, little is known about the underlying mechanisms for SO₄^2- transport in cartilaginous fish, largely due to the extraordinarily elaborate four-loop configuration of the nephron, which consists of at least 10 morphologically distinguishable segments. In the present study, we determined cDNA sequences from the kidney of holocephalan elephant fish (Callorhinchus milii) that encoded solute carrier family 26 member 1 (Slc26a1) and member 6 (Slc26a6), which are SO₄^2- transporters that are expressed in mammalian and teleost kidneys. Elephant fish Slc26a1 (cmSlc26a1) and cmSlc26a6 mRNAs were co-expressed in the proximal II (PII) segment of the nephron, which comprises the second loop in the sinus zone. Functional analyses using Xenopus oocytes and the results of immunohistochemistry revealed that cmSlc26a1 is a basolaterally-located electroneutral SO₄^2- transporter, while cmSlc26a6 is an apically-located, electrogenic Cl^-/SO₄^2- exchanger. In addition, we found that both cmSlc26a1 and cmSlc26a6 were abundantly expressed in the kidney of embryos; SO₄^2- was concentrated in a bladder-like structure of elephant fish embryos. Our results demonstrated that the PII segment of the nephron contributes to the secretion of excess SO₄^2- by the kidney of elephant fish. Possible mechanisms for SO₄^2- secretion in the PII segment are discussed.