We examined ionoregulatory characteristics of two species of characiform fish and two species of cichlids that are not native to the Rio Negro, an extremely ion-poor, acidic tributary of the Amazon River, in order to gain insight into the origin of the specializations possessed by Rio Negro fish. The two characiform species examined, Congo tetras (Phenacogrammus interruptus) and black neon tetras (Hyphessobrycon herbertaxelrodi), had high-affinity/high-capacity transporters that produce high rates of uptake in dilute water. Na+ uptake for both was pH insensitive (down to pH 3.5). Exposure to 100 μmol L-1 phenamil had no effect on Na+ uptake in either species, while exposure to 1 mmol L-1 NH4Cl- (high external ammonia [HEA]) slightly stimulated Na+ uptake in Congo tetras. Exposure to "Na+-free" water significantly inhibited Na+ uptake (by 65%-85%) but had no effect on net ammonia flux. The two cichlid species examined, convict cichlids (Amatitlania siquia) and red point cichlids (Archocentrus sp.), had high-affinity but low-capacity transporters that yield low rates of uptake in dilute media. Sodium transport was pH sensitive and completely inhibited at pH 3.5. Phenamil exposure inhibited Na+ uptake by 60% in convict cichlids but had no effect on red point cichlids, and exposure to HEA reduced Na+ uptake in both species by 70%-85%. Exposure to "Na+-free" water reduced Na+ uptake by 80%-85%, and in convict cichlids it also reduced net ammonia flux by about 50%. The ionoregulatory characteristics described for both groups are strikingly similar to those for Rio Negro species, and we suggest that they may be ancestral physiological traits for these two groups. Further, if this is the case, it seems likely that these traits existed before the Rio Negro and may explain the great success of these species-rich groups in colonizing the river despite its challenging chemistry.