The transport and the activity of many regulatory proteins is modulated by bound heparan sulfate (HS). HS ‘phage display antibodies are powerful tools for the analysis of the native HS structures in situ, but their epitopes are currently not well defined. The analysis of the measures related to access to a number of HS-specific antibodies by competitive binding assays with defined, chemically modified heparins reveals that O-sulfates are necessary for binding, an increase in sulfation is not necessarily correlated with an increase in antibody reactivity. The binding assays and immunohistochemistry showed that some antibodies can recognize different epitopes. Not simple linear sequences, but families of structurally similar motifs based on the minimal variation of sulfation and conformation influence the affinity of the interaction. The modeling of the antibody on the computer shows that they possess strong basic CDR3 regions, which define the number of possible orientations of the HS bond. Contrary to expectations, significant differences between the presence of epitopes in tissue samples that show was that the in vitro – specificity does not necessarily correlate with the specificity of in situ / vivo. The epitopes are therefore more complex than previously thought. Overall, these data points are important for structure-activity relationships of HS.