This study examines the interaction between the monofluorinated phospholipid 1-palmitoyl-2-[16-fluoropalmitoyl]sn-glycero-3-phosphocholine (F-DPPC) and the unsaturated lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). F-DPPC is a unique phospholipid which interdigitates without the use of inducing agents. Increasing concentrations of DOPC were added to F-DPPC in an effort to determine if F-DPPC remains interdigitated. Differential scanning calorimetry (DSC) was used to analyze the thermotropic phase behavior of these lipid mixtures. Introducing DOPC decreases the main transition temperature ( T m), reduces the main transition enthalpy, and increases the T m hysteresis. The high T m hysteresis implies that the membrane remains interdigitated even at high amounts of DOPC. In addition, fluorescence measurements were taken with two fluorescent probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and the DPH-labeled analogue of DPPC, 2-(3-(diphenylhexatrienyl) propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-DPH HPC). In contrast to our DSC results, the increase in fluorescence intensity suggests that as more DOPC is added, less of the F-DPPC/DOPC system is interdigitated. Our fluorescence polarization results reveal that DOPC increases the fluidity of F-DPPC membranes below the T m. From these results, it is not definitive if DOPC impedes the interdigitation of F-DPPC, although the presence of the two cis double bonds in the hydrocarbon tail of DOPC clearly disrupts the lipid packing of F-DPPC bilayers.