Methods of estimating the mesophyll conductance (g(m)) to the movement of CO(2) from the substomatal airspace to the site of fixation are expensive or rely upon numerous assumptions. It is proposed that, for C(3) species, measurement of the response of photosynthesis to [O(2)] at limiting [CO(2)], combined with a standard biochemical model of photosynthesis, can provide an estimate of g(m). This method was used to determine whether g(m) changed with [CO(2)] and with water stress in soybean leaves. The value of g(m) estimated using the O(2) response method agreed with values obtained using other methods. The g(m) was unchanged over the tested range of substomatal [CO(2)]. Water stress, which decreased stomatal conductance (g(s)) by about 80%, did not affect g(m), while the model parameter V(Cmax) was reduced by about 25%. Leaves with g(s) reduced by about 90% had g(m) values reduced by about 50%, while V(Cmax) was reduced by about 64%. It is concluded that g(m) in C(3) species can be conveniently estimated using the response of photosynthesis to [O(2)] at limiting [CO(2)], and that g(m) in soybean was much less sensitive to water stress than g(s), and was somewhat less sensitive to water stress than V(Cmax).