Saturated sphingolipids have high acyl chain order. Our aim was to study how palmitoylated sphingomyelin (PSM), ceramide (PCer), glucosyl (GlcPCer)-, and galactosylceramide (GalPCer) were able to order the bulk acyl chains of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), in comparison with cholesterol. For this reason, we used lipid probes which had preferred phases that were either the disordered phase (1-oleoyl-2-propionyl[DPH-sn-glycero-3-phosphcholine (18:1-DPH-PC) or the ordered phase (trans parinaric acid (tPA). DPH was also used, although it has no clear phase preference. We measured steady-state anisotropy (all probes) and performed fluorescence lifetime analysis (tPA) as a function of composition and temperature. At concentrations where the saturated sphingolipids were not aggregated into ordered domains (and 23 degrees C), they did not increase POPC acyl chain order as determined from 18:1-DPH-PC anisotropy. As expected, cholesterol increased the POPC acyl chain order linearly as a function of concentration (0-28 mol %). Since PCer already forms ordered domains below 5 mol % (at 23 degrees C), we measured the acyl chain ordering effect of PCer at 50 degrees C (0-13 mol %) and observed that PCer ordered POPC acyl chains as efficiently as cholesterol. We conclude that the bulk acyl chain order of POPC was not markedly affected in bilayers where disordered and ordered domains coexist.