Agonist occupancy of high affinity histamine H3 receptors on AtT-20 cells induces increased ACTH release. However, the signal transduction process by which this occurs is presently unknown. As a first step in characterizing this pathway, we have examined the effects of a variety of nucleotides and nucleotide analogs on Na-methylhistamine binding to these receptors. Nonhydrolyzable guanine nucleotide analogs inhibit up to 40% of the [3H]Na-methylhistamine binding by increasing the dissociation rate of the ligand from the receptor and, thereby, reducing receptor affinity. Pertussis toxin also decreases the affinity of the H3 receptors and ADP ribosylates a 41 kDa protein. Neither GTP gamma S nor pertussis toxin change Bmax. These data indicate that the H3 receptors on these cells are coupled to a G protein of the Gi subclass.