The intramolecular dynamics of the excimer forming dipyrenyl lipids (DipynPC) of different chain lengths (n) in ethanol and in dimyristoylphosphatidycholine (DMPC) membranes was investigated by the use of frequency-domain fluorescence intensity decay technique. Based on a 3-state model, the extent of aggregation and rotational rate of the two intralipid pyrene moieties in the dipyrenyl lipids were estimated from the frequency-domain data. In ethanol (20 degrees C), the rotational rate for DipynPC increased progressively as n was varied from 4 to 12. At the gel (L beta)-to-liquid crystalline (L alpha) phase transition of DMPC (approximately 23 degrees C), the rotational rate increased and aggregation decreased significantly for Dipy10PC, whereas only the rotational rate was changed for Dipy4PC. In the presence of 30 mol% cholesterol, significant increases in both the rotational rate and aggregation were observed for Dipy10PC in both L beta and L alpha phases. However, for the case of Dipy4PC, an increase in the rotational rate but a decrease in the aggregation were noticed only in the L beta phase, and no similar changes were detected in the L alpha phase. Our results indicate differential effects of cholesterol on the conformational dynamics of acyl chains at different depths of the membranes.