This study investigates the dynamic behavior of 1,6-diphenyl-1,3,5-hexatriene (DPH) in C(18):C(10)phosphatidylcholine [C(18):C(10)PC] bilayers. C(18):C(10)PC is an asymmetric mixed-chain phosphatidylcholine known to form mixed-interdigitated structures below the transition temperature and form partially interdigitated bilayers above the transition temperature. The rotation of DPH in C(18):C(10)PC has been described in terms of the thermal coefficient of rotation using the modified Y-plot method which takes into account the limiting anisotropy value. During the phase transition of C(18):C(10)PC, DPH exhibits a thermal coefficient b2M = 0.41 - 0.51 degrees C-1 which is similar to the b2M values obtained with noninterdigitated phosphatidylcholine bilayers. Differential polarized phase-modulation fluorometry has also been employed to study the dynamic behavior of DPH in C(18):C(10)PC in real time. The data show that DPH contains considerable motion in the highly ordered mixed interdigitated bilayers. The DPH motion steadily increases with an increase in temperature as shown by the rotational correlation time, and the wobbling diffusion constant. However, the limiting anisotropy, the order parameter, and the width of the lifetime distribution undergo an abrupt decrease, and a corresponding abrupt increase in the cone angle, at approximately 16 degrees C. This temperature range is near the onset temperature of the phase transition as determined by differential scanning calorimetry. The rotational parameters show strong hysteresis on heating and cooling. All the rotational parameters derived from DPH fluorescence in mixed interdigitated C(18):C(10)PC exhibit magnitudes similar to those obtained from non interdigitated gel phases of symmetric diacylphosphatidylcholines. These results, combined with those obtained with dehydroergosterol (Kao, Y. L., P. L.-G. Chong, and C. Huang. 1990. Biochemistry. 29:1315-1322), suggest that considerable rotational mobility of small molecules can be sustained in an intramolecularly highly ordered interdigitated lipid matrix, implying that the membrane maintains a fluid environment around membrane perturbants even when the lipid matrix is extensively interdigitated.