Pro- and eukaryotic cells express multiple GTP-binding proteins that play crucial roles in signal transduction. GTP-binding proteins possess a highly conserved NKX D motif critically involved in guanine binding. In order to selectively activate a defined GTP-binding protein, base-specificity can be switched from guanine to xanthine by mutating the conserved aspartate into asparagine (D/N-mutation). This approach was very successful at elucidating the function of structurally diverse GTP-binding proteins in complex systems. However, attempts to generate functional xanthine nucleotide-specific alpha-subunits of heterotrimeric GTP-binding proteins (G-proteins) met more difficulties. Recent studies have shown that a sufficiently high GDP-affinity is critical for functional expression of xanthine nucleotide-selective G-protein mutants. Moreover, xanthosine 5'-[gamma-thio]triphosphate and xanthosine 5'-[gamma, beta-imido]triphosphate are not functionally equivalent activators of D/N-G-protein mutants. We are now in the position to exploit xanthine nucleotide-specific G-proteins to dissect signaling pathways activated by a given G-protein in complex systems.