Recent studies have shown that increases in yield and heat resistance in successively released, commercial lines of Pima cotton (Gossypium barbadense L.) are positively correlated with increases in stomatal conductance. In the present study, we used a recently developed method to isolate guard cells from cotton leaves to compare guard cell properties in low and high yielding Pima lines. Measurements of oxygen consumption of guard cell suspensions showed that guard cells from a high-yielding, high-conductance Pima line had higher respiration rates than guard cells from a low-yielding, low-conductance line. A third, intermediate line had guard cells with intermediate rates of oxygen consumption. Inhibition of oxygen consumption by KCN and sodium azide implicated oxidative phosphorylation. Rates of CCCP-sensitive proton extrusion, measured as medium acidification of the guard cell suspensions, were also higher in the high-yielding, high-conductance line. The temperature-dependence of respiration and proton extrusion paralleled the temperature-dependence of stomatal conductance in the intact leaves. These results indicate that the conductance increases ensuing from selection for higher yields and heat resistance in Pima cotton are associated with modifications of some key guard cell properties.