AbstractWe have investigated the relationship between the spreading of anchorage‐dependent cells and the surface‐density distribution of plasma membrane adhesion proteins. The surface positioning and density of integrin β1, caveolin‐1 (cav‐1), the phosphorylated caveolin‐1 (p‐cav‐1) and the focal adhesion kinase (FAK) located on the adhering cell membrane (ACM) of HUVEC cells was studied. Imaging with TIRF microscopy was used, which enabled us to observe a few‐nanometers‐thin section of the cell above the plasma membrane in combination with image‐based analyses. Integrin β1 and cav‐1 have spatial interdependence on the ACM. Cells treated with substances that act on cell spreading caused changes in the size of the ACM area, as well as a redistribution of several proteins under investigation. Changes to the ACM area correlated positively with those to the surface density of the cav‐1. The high integrin β1 and the low cav‐1 surface density, and vice versa, following the treatments show that the presence of one of them not only spatially excludes, but also reduces, the occurrence of the other protein on the ACM, which indicates a regulative mechanism between integrin β1 and cav‐1.