Mono-ADP-ribosylation is a reversible, post-translational modification of cellular proteins that has been implicated in regulation of signal transduction, muscle cell differentiation, and protein trafficking and secretion. The reaction is catalysed by mono-ADP-ribosyltransferases that transfer a single ADP-ribose moiety from p-NAD⁺ to a specific amino-acid of acceptor proteins. An ADP-ribosylation reaction occurs in intact cells on the p subunit of heterotrimeric G proteins that is carried out by an arginine- specific, plasma-membrane-associated, mono-ADP-ribosyltransferase. This modification is reversed by a cytosolic ADP-ribosylhydrolase that regenerates native Py dimer by releasing the bound ADP-ribose. Once ADP-ribosylated, the py dimer is inactive towards its effector enzymes, such as adenylyl cyclase, phosphoinositide 3-kinase and phospholipase C. It thus appears that endogenous P subunit mono-ADP-ribosylation might represent a novel cellular mechanism for the modulation of the G-protein-mediated signal transduction machinery through a direct regulation of the py dimer. In this study, the mechanisms that regulate endogenous mono-ADP-ribosylation of the p subunit have been investigated. The reaction appears to be under hormonal control both in vitro and in vivo, since the levels of ADP-ribosylated p are increased upon activation of certain G-protein-coupled receptors (GPCRs), such as thrombin, serotonin and cholecystokinin receptors. Conversely, hormonal stimulation by additional GPCRs, such as the GnRH receptor, can lead to a decrease in p subunit mono-ADP-ribosylation. Thus, ADP-ribosylation of the py dimer can be differentially regulated by different GPCRs in a receptor-type-dependent manner. In addition, the involvement of the ADP-ribosylating factor ARF6 in GnRH-mediated regulation of p subunit mono-ADP-ribosylation is demonstrated. Indeed, removal of ARF6 from plasma membranes results in loss of GnRH-mediated inhibition of p subunit mono-ADP-ribosylation, which can be fully restored by re-addition of purified ARF6. In conclusion, the results reported in this thesis allow the definition of the mechanisms that regulated endogenous ADP-ribosylation of the p subunit, and demonstrate a novel role for ARF6 in hormonal regulation of p subunit mono-ADP-ribosylation.