In the present series of papers, we describe the results of a systematic study on the anchoring of cholesterol-tagged oligonucleotides to phospholipids hi layers followed by membrane-assisted hybridization of the complementary strand in solution. This paper describes the anchoring of novel cholesterol-modified DNA-18mers in supported lipid bilayers (SLB) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine: we compared the behavior of two anchoring functionalities differing in the number of cholesterol units and in the length of a spacer group. Quartz Crystal Microbalance with impedance monitoring (QCM-Z) measurements showed that both oligonucleotides insert into the bilayer membrane through cholesterol anchoring: however, dramatic differences, in terms of surface organization and thickness, arc found as the number of anchoring units increases. In the case of multiple cholesterol units, a peculiar three-regimes concentration dependence was revealed and correlated to the effective size of the adsorbing units. Interestingly, for high oligonucleotide concentration, the adsorption process was rationalized in terms of a compaction model of amphiphilic DNA molecules. QCM-Z temperature cycles of the SLB-anchored double strands provided clear evidence for reversible hybridization at the bilayer interface.