The chromatographic capacity factors of 39 neutral and basic compounds were measured on an immobilized artificial membrane—phosphatidylcholine—drug discovery (IAM–PC–DD) HPLC column, and the values compared with both octanol/water partition coefficients and capacity factors previously obtained on an IAM–PC–MG column. These two columns differ in their lipidic phase, since the IAM–PC–MG phase is made of phosphatidylcholine as found in biomembranes, whereas the glycerol linker is absent in the IAM–PC–DD phase. We found that the two phases interact differently with basic compounds at different degrees of ionization. On the IAM–PC–MG column, ionized compounds are as strongly or more strongly retained than isolipophilic neutral compounds. In contrast, their retention on the IAM–PC–DD column is less strong than, or at most as strong as, that of isolipophilic neutral compounds. The IAM–PC–MG data appear as better predictors of the interactions between drugs and biological membranes. Indeed, they correlate better than the IAM–PC–DD data with partitioning in both biological membrane and liposomes; moreover, they are better correlated with biological activities from the literature. These results suggest that even modest modifications in the structure of IAM phospholipids can have a major effect on the retention of basic compounds. We conclude that an acceptable IAM–HPLC estimate of the interactions between biomembranes and basic compounds should rely on stationary phases that reproduce the structure of natural phospholipids.