The degree to which cell membranes are barriers to CO2 transport remains controversial. Proteins, such as aquaporins and Rh-complex, have been proposed to facilitate CO2 transport but this implies that the non-channel component of membranes must have greatly reduced CO2 permeability; otherwise, permeation across the lipid matrix would short-circuit the channel-mediated pathway. However, other studies have demonstrated that CO2 crosses lipid bilayers very rapidly. To determine whether membrane CO2 permeation is rate-limiting for gas transport, the spread of CO2 across multicellular tissue-growths (spheroids) was measured using intracellular pH (pHi) as a spatial read-out. Experimental data were analysed using a carefully parameterized diffusion-reaction algorithm to extract information about CO2 dynamics. Although H+-yielding CO2 hydration is rate-limited by intracellular carbonic anhydrase (CA) activity locally, time-delays in pHi-changes at different spheroid-depths report the spread of CO2. These delays, readily resolvable in spheroids, can ascertain whether CO2 diffuses freely or is restricted by membrane permeation. Experiments were performed in the presence of the membrane-impermeant CA inhibitor 4-aminomethyl benzenesulfonamide (30 µM) to eliminate CA-facilitated CO2 diffusion.Colorectal HCT116 cells have basal permeability to water (Pf=25±1 µm/s; measured by hypo-osmotic dilution of intracellular calcein fluorescence) and NH3 (Pm,NH3=147±8 µm/s; measured from changes in pH-sensitive cSNARF1 fluorescence evoked by ultra-rapid (<20 ms) addition/removal of NH3/NH4+). Furthermore, Pf and Pm,NH3 were unaffected by blockers of aquaporins and Rh-complex (1 mM Hg2+, 2 mM p-chloromercuribenzoic acid [PCMB], 30 µM 4,4′-diisothiocyano-2,2′-stilbene-disulfonic acid [DIDS]), indicating the functional absence of aquaporins and NH3 gas channels. Despite the absence of protein-facilitated water and NH3 permeation, CO2 diffusivity in HCT116 spheroids was fast: only 24±4% lower than in pure water, which can be accounted for fully by volume-exclusion due to proteins (75 g/L). Diffusivity was unaffected by PCMB and DIDS, but reduced under hypertonic conditions (adding 300 mOsm mannitol) which increases intracellular protein-crowding. Comparably high CO2 diffusivity measurements (~25% slower than in pure water) were obtained in spheroids of T47D breast cells (basal water permeability; Pf=20±1 µm/s) and NHDF-Ad fibroblasts (aquaporin 1 and 9-facilitated water permeability; Pf=62±5 µm/s). In contrast, diffusivity of NH3, a smaller but less lipophilic gas, was considerably slower than in pure water (~75%), as expected from rate-limiting membrane permeation. In conclusion, membranes, even in the functional absence of proposed gas channels, do not restrict CO2 venting from tissue-growths.