Atomic force microscopy (AFM) measurements have been applied to the study of how the nature of the acyl chains of sphingomyelin (SM) influence its lateral distribution in the ternary lipid mixture made of SM/cholesterol/ 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The study is focused on the importance of the hydrophobic part of the SM molecule for domain formation. AFM-force spectroscopy measurements showed that the presence of the double bound in the 24:1 SM molecule, in mixtures with cholesterol (CHO) or in pure bilayers, led to a decrease in the molecular packing. Confocal and AFM showed, at meso and nanoscale respectively, that unlike 16:0 and 24:0 SM 24:1 SM does not induce phase segregation in ternary lipid mixtures with DOPC and CHO. This ternary lipid mixture had a nanomechanical stability intermediate between those displayed by liquid-ordered (Lo) and liquid-disordered (Ld) phases, as reported by AFM-force spectroscopy measurements, evidencing that 24:1 SM is able to accommodate both DOPC and CHO, forming a single phase. Confocal experiments on giant unilamellar vesicles made of human, sheep and rabbit erythrocyte ghosts rich in 24:1 SM and CHO, showed no lateral domain segregation. This study provides insights on how the specific molecular structure of SM affects the lateral behavior and the physical properties of both model and natural membranes. Specially the data suggest that unsaturated SM may help in keeping membrane lipids in a homogeneous mixture, rather than in separated domains.