The perturbations induced by second messenger diacylglycerols (DAGs) into bovine brain phosphatidylcholine (BBPC) bilayers in the presence or absence of bovine brain sphingomyelin (SM) and/or cholesterol were studied by H-2 NMR. Addition of 15 mol% DAG to BBPC bilayers did not induce non-bilayer lipid phases in the temperature range 30-60degreesC. Similar measurements performed in the presence of cholesterol revealed that cholesterol progressively destabilizes PC bilayers with respect to DAG-induced perturbations. Thus, at 40 mol% cholesterol, addition of 15 mol% DAG induced the formation of non-bilayer (isotropic and inverted hexagonal) phases at 60degreesC. Whereas some lateral separation of the bilayers into domains of different cholesterol contents was observed in BBPC/cholesterol membranes, such a lateral heterogeneity was greatly facilitated by the addition of SM. Since both a tendency to form non-bilayer lipid phases and lateral heterogeneity of the membranes are associated with increased activation of a number of membrane-associated enzymes, our results suggest that SM- and cholesterol-enriched regions of biological membranes (rafts) provide an environment with increased sensitivity to the generation of lipid second messengers and modified transmembrane signal transduction properties. (C) 2002 Elsevier Science (USA). All rights reserved.