Methods of estimating the mesophyll conductance (gm) to the movement of CO2 from the substomatal airspace to the site of fixation are expensive or rely upon numerous assumptions. It is proposed that, for C3 species, measurement of the response of photosynthesis to [O2] at limiting [CO2], combined with a standard biochemical model of photosynthesis, can provide an estimate of gm. This method was used to determine whether gm changed with [CO2] and with water stress in soybean leaves. The value of gm estimated using the O2 response method agreed with values obtained using other methods. The gm was unchanged over the tested range of substomatal [CO2]. Water stress, which decreased stomatal conductance (gs) by about 80%, did not affect gm, while the model parameter VCmax was reduced by about 25%. Leaves with gs reduced by about 90% had gm values reduced by about 50%, while VCmax was reduced by about 64%. It is concluded that gm in C3 species can be conveniently estimated using the response of photosynthesis to [O2] at limiting [CO2], and that gm in soybean was much less sensitive to water stress than gs, and was somewhat less sensitive to water stress than VCmax.