Crystallographic structures of wild-type and mutant NOS isoforms complexed with substrate, intermediate, inhibitor, cofactor, and cofactor analogs are currently available. However, because of the high level of amino-acid conservation and the consequent similarity in dimeric quaternary structure as well as in the active site of NOS isoforms, structure-based isoform-selective inhibitor design is still a very challenging task. Nevertheless, the comprehension of the structural determinants for selectivity among the isoforms is fundamental for the design of further potent and more selective inhibitors. Computational techniques, based on the knowledge of the tridimensional structure of the isozymes, have been already applied to understand the significant isoform selectivity shown by some compounds. Collectively these structure-based approaches, in combination with SAR studies, have been able to explain the structural reasons of this selectivity.