Pertussis toxin catalyzes the ADP-ribosylation of the inhibitory subunit (Ni) of adenylate cyclase. Despite several studies which demonstrate that pertussis toxin influences cyclic AMP accumulation and hormone secretion in normal anterior pituitary cells, the target protein(s) for this toxin in these cells has not been identified. We have examined pertussis toxin mediated ADP-ribosylation in membrane preparations of tumor-derived (235-1, GH4C1, GH3) and normal anterior pituitary cells. Autoradiograms of SDS gels reveal that in the presence of [32P]NAD and pertussis toxin, a 40 kilodalton protein band was labeled in membrane preparations from cells cultured with vehicle. Such labeling was diminished when the cells were exposed to pertussis toxin (35 ng/ml) for 18 hours. Similar results were found in both tumor-derived and normal (monkey and rat) anterior pituitary cells. The pertussis toxin specific band was further resolved into two bands of approximately 39 and 41 kilodaltons. Autoradiograms of two dimensional gels revealed two ADP-ribosylated spots with isoelectric points of 5.7 and 6.2, although the molecular weights appeared identical (approx. 40 kilodaltons). Cholera toxin, which catalyzes the ADP-ribosylation of a 45 kilodalton protein did not prevent labeling of the pertussis toxin-specific band(s) in cells pretreated with cholera toxin. These results suggest that pertussis toxin specifically mediates ADP-ribosylation of the Ni protein in normal anterior pituitary cells.