Neuronal Nitric Oxide synthase, unlike its endothelial and inducible counterparts, displays a PDZ (PSD-95/Dlg/ZO-1) domain located at its N-terminus involved in subcellular targeting. The C-termini of various cellular proteins insert within the binding groove of this PDZ domain and determine the subcellular distribution of nNOS. The molecular mechanisms underlying these interactions are poorly understood since the PDZ domain of nNOS can apparently exhibit class I, class II and class III binding specificity. In addition, it has been recently suggested that the PDZ domain of nNOS binds with very low affinity to the C-termini of target proteins and a necessary simultaneous lateral interaction must take place for binding to occur. We describe herein that the PDZ domain of nNOS can behave as a bona fide class III PDZ domain and bind to C-terminal sequences with acidic residues at the P-2 position with low micromolar binding constants. Binding to C-terminal sequences with a hydrophobic residue at the P-2 position plus an acidic residue at the P-3 position (class II binding) can also occur, although interactions involving residues extending up to the P-7 position mediate this type of binding. This promiscuous behavior also extends to its association to class I sequences, which must display a Glu residue at P-3 and a Thr residue at P-2. By means of site directed mutagenesis and NMR spectroscopy we have been able to identify the residues involved in each specific type of binding and rationalize the mechanisms used to recognize binding partners. Finally, we have analyzed the high-affinity association of the PDZ domain of nNOS to claudin-3 and claudin-14, two tight-junction tetraspan membrane proteins that are essential components of the paracellular barrier.