Camptothecins are potent antitumor agents that stabilize the covalent binding of topoisomerase I to DNA forming a reversible ternary complex which, following collision with the replication forks, converts the single-strand breaks into lethal double-strand breaks. This cytotoxic mechanism has been originally ascribed to the closed lactone form, because opening of the lactone ring resulted in loss of antitumor activity. Since the lipophilic lactone favours passive diffusion into the cancer cells, the stability of the closed form is expected to be predictive for activity. Thus, the in vivo pharmacological behavior of camptothecins, which is dependent on the pH-dependent dynamics, is likely a critical determinant of their antitumor efficacy and therapeutic index. The physicochemical properties could influence a number of cellular and in vivo interactions, including stability of the ternary DNA-enzyme-drug complex, binding to serum proteins, recognition by transport systems. These interactions are also implicated in the processes responsible of toxic side effects and drug resistance which are major limitations of the efficacy of camptothecin-based therapy. A number of strategies have been developed to overcome the limitations associated with the peculiar in vivo reactivity and the reversibility of drug-target interaction. Modifications with hydrophilic or lipophilic substituents at specific positions may have a variable (and somewhat opposite) influence on interaction with the intracellular target and plasma proteins and on recognition by membrane transporters. Here, we highlight the interactions of camptothecins which could be exploited to optimize therapeutic efficacy.