Phosphoinositide content was measured in erythrocyte membranes from 11 patients with cystic fibrosis (CF) and from 12 control subjects to determine whether altered levels of phosphatidylinositol-4-phosphate (Ptdlns4P) or phosphatidylinositol-4,5-bisphosphate (Ptdlns(4,5)P2) are responsible for the decrease in Ca2+-adenosine triphosphatase (Ca2+-ATPase) activity in this disorder. Isolated membranes were extracted with an acidified chloroform-methanol solvent system. The recovered lipids were separated by one-dimensional thin-layer chromatography and quantified with a colorimetric assay for phosphorus. The results are expressed in molar percent, moles of phosphoinositide times 100 divided by the total number of moles of phospholipid per membrane. The means +/- SEM of Ptdlns(4,5)P2, Ptdlns4P, and phosphatidylinositol (Ptdlns) in CF membranes (1.07 +/- 0.18, 1.02 +/- 0.22, and 2.32 +/- 0.36 molar percent, respectively) were indistinguishable from controls (0.91 +/- 0.14, 0.85 +/- 0.12, and 2.21 +/- 0.32 molar percent, respectively) (P greater than 0.20 for all three pairs). The accuracy of quantitative recovery throughout the procedure was determined by adding a radioactive internal standard, L-3-phosphatidyl[2-3H]inositol to 10 membrane preparations. Although quantitative recoveries, as determined by percent radioactivity recovered, varied from 54% to 92%, mean Ptdlns(4,5)P2, Ptdlns4P, and Ptdlns levels appropriately corrected from tracer loss were still indistinguishable between the two groups. We conclude that absolute phosphoinositide levels are not altered in cystic fibrosis erythrocyte membranes and that the differences in Ca2+-ATPase activity cannot be explained on this basis.