Summary Medium acidification by cell cultures from soybean ( Glycine max. L.) was stimulated by the ether phospholipid platelet-activating factor (PAF), 1-O-alkyl-2-acetyl- sn -glycero-3-phosphocholine. Acidification was dependent on the concentration of PAF, on the cell density and on the presence of calcium ions in the medium. Ethyleneglycol-bis(aminoethylether)-N,N,N〲,N〲-tetraacetic acid (EGTA) equaling the calcium concentration reduced the proton extrusion whereas addition of the calcium ionophore A 23187 increased it. Cells in the logarithmic growth phase 3—4 days after inoculation, showed the strongest response to the lipid. In vitro PAF had a stimulatory effect on the ATPase activity in both the tonoplast-and the plasma membrane-enriched fraction. In microsomes isolated from cultured soybean cells PAF stimulated the phosphorylation of a 55 kDa, a 97 kDa and a 120 kDa polypeptide. With an antibody prepared against a cytosolic fragment of the plasma membrane H + -ATPase an immunostaining signal was obtained at 120 kDa, suggesting that the phosphoprotein having the same molecular mass could be the plasma membrane H + -ATPase. After membrane fractionation by a sucrose gradient the 55 kDa and the 97 kDa phosphoproteins were found predominantly in fractions of low density correlating with markers of either tonoplast or endoplasmatic reticulum.