The fluidity of the plasma membrane is thought to affect the responsiveness of blood platelets. We measured membrane fluidity in a single cell by Fluorescence Recovery after Photobleaching (FRAP) of the lipophilic probe DiIC14. Since platelets are too small for this technique, we used the human megakaryoblastic cell-line MEG-01, which shares many properties with platelets. MEG-01 cells were cultured for 44 h with simvastatin or mevalonate to change the cholesterol content, enabling analysis of signal processing at cholesterol/phospholipid ratios (C/P) between 0.20 and 0.31. The diffusion of DiIC14 correlated inversely with the C/P ratio with lateral diffusion coefficients (D) of 3.28 x 10(-9) cm2/s at a low C/P decreasing to 2.55 x 10(-9) cm2/s at a high C/P ratio. The mobile fraction was 65% and constant at the different C/P ratios. The relation between lipid diffusion and signal processing was measured following stimulation with 10 U/ml thrombin at 22 degrees C. There were only little differences in phosphatidylinositol metabolism, Ca2+ influx or mobilization and prostaglandin I2-induced formation of cyclic AMP. At 37 degrees C, cells with a high C/P ratio showed increased phosphatidylinositol metabolism, but these differences had no major effect on the Ca2+ responses. These data demonstrate that in megakaryoblasts the lateral diffusion of lipids is inversely correlated with the C/P ratio, but within the range of 0.20-0.31 the influence on signal processing is minor.