Although alterations in fatty acid composition of phospholipids in plasma membranes had no effect on activities of plasma membrane ATPases of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae cells grown in the absence of ethanol (basal enzymes), they significantly affected the susceptibilities of the enzymes to in vivo activation induced by ethanol: the maximal values for the activated enzymes in cells pregrown with 0.6 mmol/L palmitic, linoleic or linolenic acid respectively were 3.6, 1.5 and 1.2-fold higher than their respective basal levels (in cells grown without ethanol), whereas the corresponding value for cells pregrown in the absence of fatty acid was 2.3-fold, with the concentrations of ethanol for the above maximal in vivo activation of enzymes being 7%, 6%, 6% and 7% (V/V) respectively. The Km values for ATP, the pH profiles, and the sensitivities to orthovanadate of the basal and the activated plasma membrane ATPases were essentially identical; however, the v(max) values of activated enzymes increased significantly. It was found that the characteristics of phospholipid fatty acid composition of plasma membrane leading to the enhanced ethanol tolerance of this strain, were also efficacious to increase the percentage of activation of plasma membrane ATPase per unit of ethanol. These data support a close correlation between the ethanol tolerance of this strain and the sensitivity of its plasma membrane ATPase to the in vivo ethanol-induced activation.