Pacific hagfish (Eptatretus stoutii; 100–400 g) were exposed to seawater equilibrated with approximately 10, 30 and 50 mm Hg pCO2 and then sampled either a) immediately after transfer, time 0, or b) after 3, 6, 12, 24, 48 (only 30 and 50 mm Hg pCO2) or 96 (only 50 mm Hg pCO2) h of exposure to elevated pCO2. Blood was obtained from anaesthetized animals for pH, hematocrit, haemoglobin, and mean cell haemoglobin concentration (MCHC) as previously described29. Plasma total CO2 (TCO2), plasma ion composition, and RBC pHi were also measured29. Tissue pHi was measured from frozen tissues using the metabolic inhibitor method29. Non-bicarbonate whole blood buffer capacity and tissue non-bicarbonate buffer capacity was determined as described previously29, calculated from the slope of Δ[HCO3−] ΔpH−1, and then expressed in mmol HCO3− pH−1 l−1 of blood or kg−1 of intracellular tissue water, over an in vivo relevant pH range. Data are presented as mean ± SEM (n = 8 in all cases except one, where n = 7). All data was analyzed for normality and equal variance before statistical analysis. Statistical differences were detected using a one-way ANOVA and, when necessary, a post-hoc Dunnett’s test. All statistical analyses were conducted using SigmaStat for Windows 3.5.0.54 (Systat Software, Inc., 2006), and all analyses were 2-tailed and interpreted using α = 0.05 to determine statistical significance.