Binary membranes composed of dimyristoylphosphatidylcholine (DMPC) and natural ceramide (up to a mole fraction Xcer = 0.25) were investigated by measuring the excimer:monomer fluorescence emission intensity ratio IE:IM for the pyrene labeled phospholipid probe 1-palmitoyl-2[(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC), by monitoring fluorescence polarization of diphenylhexatriene (DPH), as well as using differential scanning calorimetry (DSC). Increasing Xcer > 0.10 both below and above the main transition temperature Tm increased IE:IM for PPDPC maximally approximately 1.6-fold at Xcer = 0.25. Above Tm, and when Xcer approaches 0.10, fluorescence polarization P for DPH increases steeply, reflecting an overall decrease in acyl chain motions. At Xcer = 0.10 there is a discontinuity in P and upon further increase in the content of ceramide a smaller, yet significant increase in P is evident. DSC revealed ceramide to increase in the pretransition temperature until at Xcer exceeding 0.07 this transition was no longer evident. Simultaneously, increasing Xcer up to 0.05 increased Tm from 23.9 to 24.6 degrees C. Total enthalpy delta Hm of the main transition diminished progressively upon increase in Xcer up to approximately 0.10. Above this concentration of ceramide a new endotherm became evident at 22.5 degrees C, and exceeding Xcer = 0.14 this endotherm became dominant. Our results indicate an enrichment of the pyrene labeled phospholipid analog into microdomains concomitant with the formation of a distinct ceramide-enriched phase at Xcer > 0.10.