In osmoregulating teleost fish, urea is a minor nitrogen excretory product, whereas in osmoconforming marine elasmobranchs it serves as the major tissue organic solute and is retained at relatively high concentrations (~400 mmol L^-1). We tested the hypothesis that urea transport across liver mitochondria is carrier-mediated in both teleost and elasmobranch fishes. Intact liver mitochondria in rainbow trout (Oncorhynchus mykiss) demonstrated two components of urea uptake, a linear component at high concentrations and a phloretin-sensitive saturable component (K_m=0.58 mmol L^-1; Vmax = 0.12 µmol h^-1 mg protein^-1) at lower urea concentrations (< 5 mmol L^-1). Similarly, analysis of urea uptake in mitochondria from the little skate (Raja erinacea) revealed phloretin-sensitive saturable transport (K_m=0.34 mmol L^-1; maximal velocity = 0.054 µmol h^-1 mg protein^-1) at low urea concentrations (< 5 mmol L^-1). Surprisingly, urea transport in skate, but not trout, was sensitive to a variety of classic ionophores and respiration inhibitors, suggesting cation sensitivity. Hence, urea transport was measured in the reverse direction using submitochondrial particles (SMPs) in skate. Transport kinetics, inhibitor response, and pH sensitivity were very similar in skate SMPs (K_m=0.65 mmol L^-1, maximal velocity = 0.058 µmol h^-1 mg protein^-1) relative to intact mitochondria. We conclude that urea influx and efflux in skate mitochondria is dependent, in part, on a bidirectional proton-sensitive mechanism similar to bacterial urea transporters and reminiscent of their ancestral origins. Rapid equilibration of urea across the mitochondrial membrane may be vital for cell osmoregulation (elasmobranch) or nitrogen waste excretion (teleost).