Nowadays there is growing awareness that the translation of the increasing number of lead compounds into clinical candidates is still a slow and often inefficient process. In order to facilitate the lead optimization procedure, due consideration must be given to the use of the right bioisosteric replacements. Very recently, we reported that exploring a chemical space of binding sites is a more effective strategy for studying the bioisosteric relationships existing among functional groups. As a continuation of our work in this field, we report herein the construction of a chemical space covered by binding sites of small molecules containing diverse amine and amidine groups. The analysis of the differences in some properties of the binding sites of these functional groups allow for gaining insights into the binding modes of positively charged groups. In addition, this study pinpoints that different types of interactions and bioisosteric relationships exist among primary, secondary, tertiary, quaternary amine, and amidine moieties.