Earlier studies have shown that the heparan sulfate (HS) on the cell surface acts as a receptor for herpes simplex virus (HSV). We have recently shown that bovine lactoferricin (LfcinB), a small part of the milk protein lactoferrin, inhibits HSV-1 and HSV-2 infection, probably by blocking the entry of the virus. The human homologue (18-42), which shares 36% sequence similarity with LfcinB (17-41), displayed much lower antiviral activity. In the present study, a set of cyclic and linear human and bovine Lfcin derivatives were constructed to investigate the relation between their affinity to HS and chondroitin sulfate (CS) and their antiviral activity against HSV-1 and HSV-2. The lactoferrin (LF) proteins and several of the Lfcin derivatives exhibited similar affinity for HS, but the LF proteins possess a much higher antiviral activity than the smaller peptides. Our structure-activity relationship studies on the Lfcin derivates confirmed that affinity for HS, that was correlated to the net positive charge, is an important factor, but does not well predict the antiviral activity. Structural parameters such as hydrophobicity, molecular size, spatial distribution of charged and lipophilic amino acids, and the cyclic structure of Lfcin also seem to be important factors to govern antiviral activity against HSV.