Plasma membranes of high purity, as assayed by marker enzymes, were obtained by phase partitioning from stele and cortex tissues of Zea mays (cv LG11) roots. ATP hydrolytic activities in the stele and cortex plasma membrane fractions were inhibited by vanadate, SW26 and erythrosin B, but insensitive to nitrate. Activity in both fractions exhibited a marked pH optimum of 6.5 and displayed typical Michelis-Menten kinetics. A high substrate specificity was apparent in both the stele and cortex plasma membrane fractions, while the lower fractions, after phase partitioning, showed less specificity for nucleotide substrates. Specific activities of the stele (67.8 jimol Pi mg"1 h~l) and cortex (78.4 fimol Pi mg-1 h-1) plasma membrane H+-ATPase were found to be very similar. Proton pumping activities in microsomal membrane fractions from stele and cortex were inhibited by nitrate and insensitive to vanadate. Ho-mogenization of stele and cortex tissue in the presence of 250 mol m~s KI resulted in microsomal fractions exhibiting vanadate-sensitive, nitrate-insensitive proton pumping activity, suggesting a plasma membrane origin for this activity. SW26 was also an effective inhibitor of proton pumping activity, although results indicated an interaction between SW26 and the fluorescent probes quinacrine and acridinc orange. Xylem perfusion of intact maize roots with various inhibitors of the plasma membrane H+-ATPase decreased the roots' ability to regulate the pH of the xylem sap, while perfusion with fusicoccin initiated a marked decrease in the pH of the xylem sap. The effects of fixatives routinely used in histochemical studies of ATPase activity were also examined. ATPase activity in both microsomal and plasma membrane fractions was inhibited by the fixatives glutaraldehyde and formaldehyde. Addition of ATP during the incubation step or during the fixation procedure decreased the degree of ATPase inhibition suggesting a substrate protection mechanism in operation. Analysis of the lipid and sterol content of the stele and cortex plasma membranes revealed that the major sterol present in both was stigmasterol which was found in significantly higher quantities in the cortex (27.4 /ig~l compared to 17.4 fig, mg"1), the other sterols detected were cholesterol, campesterol and sitosterol. The major lipids were phosphatidylcholine (PC) and phosphatidylethanolamine (PE) with PC more abundant in the stele (88.9 /ig mg~r) than in the cortex (59.0 fig mg"1); levels of PE were similar in both tissues.