The heterotrimeric G protein subunit G alpha i can be activated by G protein-coupled receptors and the cytosolic protein Ric-8A, the latter of which is also known to prevent ubiquitin-dependent degradation of Gai. Here we show that the amounts of the three G alpha i-related proteins G alpha i1, G alpha it, and G alpha i3, but not that of G alpha q, are rapidly decreased by cell treatment with pertussis toxin (PTX). The decrease appears to be due to ADP-ribosylation of Gai, because PTX treatment does not affect the amount of a mutant G alpha i2 carrying alanine substitution for Cys352, the residue that is ADP-ribosylated by the toxin. The presence of endogenous and exogenous Ric-8A increases G alpha i stability as shown in cells treated with the protein synthesis inhibitor cycloheximide; however, Ric-8A fails to efficiently stabilize ADP-ribosylated G alpha i. The failure agrees with the inability of Ric-8A to bind to ADP-ribosylated G alpha i both in vitro and in vivo. Thus PTX appears to exert its pathological effects at least in part by converting G alpha i to an unstable ADP-ribosylated form, in addition to the well-known inability of ADP-ribosylated G alpha i to transduce signals triggered by G protein-coupled receptors. (C) 2017 Elsevier Inc. All rights reserved.