CO2 transport properties of blood were examined in the lamprey Petromyzon marinus and the hagfish Myxine glutinosa. In order to evaluate possible chloride/bicarbonate exchange limitations, experiments were conducted under control conditions and in the presence of an ionophore to permit equilibrium distribution of chloride, bicarbonate, and protons across the erythrocyte membrane. The ionophore, tri-n-propyl tin chloride, markedly altered the CO2 transport properties and apparent nonbicarbonate buffering characteristics of the blood of Petromyzon marinus. In addition, the distributions of protons, bicarbonate and chloride ions across the erythrocyte membrane were very different from each other under control conditions, but became very similar in the presence of the anionic ionophore. The CO2 transport properties of the blood of Myxine glutinosa were not significantly different in the presence of the ionophore. Small but significant changes were observed, however, in erythrocyte pH, chloride concentration and water content in the presence of tri-n-propyl tin chloride. These results demonstrate that chloride/bicarbonate exchange limitations and possibly active transport of protons contribute to the unique CO2 transport properties in the blood of the lamprey, Petromyzon marinus. In the hagfish, Myxine glutinosa, the importance of anion exchange limitations or active proton transport with regard to the CO2 carrying properties of the blood are clearly much less than in the lamprey under the in vitro conditions of this study.