Mutations and altered functionality of the inhibitory subfamily of G proteins (Gi) are involved in pathological states. Compounds able to activate Gi in a receptor-independent manner would be useful to treat these pathological conditions. Aimed to study Gi direct activation we have reconstituted a recombinant transductor-effector complex cloning both the mammalian Galpha(i1) subunit and adenylate cyclase (AC). The myristoylation of Galpha, fundamental for interaction with AC, was obtained in the procaryotic expression host Escherichia coli transformed with a single plasmid containing both the coding sequences for human Galpha(i1) and Saccharomyces cerevisiae myristoyl transferase. AC-V isoform was obtained by the expression of its cytosolic domains. A recent synthesized molecule, named BC5, was tested to evaluate its pharmacological profile in a Gi/AC cell-free complex model. In this functional transductor-effector system BC5 was able to activate Gi signalling, moreover providing a new tool to give a better insight into G-protein receptor-independent modulation.