A DNA, cloned after screening a rat genomic bank with probes derived from the sequence of a putative dog histamine H2 receptor [Gantz, I., Schäffer, M., Delvalle, J., Logsdon, C., Campbell, V., Uhler, M. & Yamada, T. (1991) Proc. Natl. Acad. Sci. USA 88, 429-433], was used to prepare a probe for Northern blot analysis and to transfect Chinese hamster ovary (CHO) cells. Distribution of the gene transcripts in guinea pig tissues was consistent with that of H2 receptors. Transfected CHO cells expressed a high density of sites binding [125I]iodoaminopotentidine, a selective H2-receptor ligand. These sites were characterized as typical H2 receptors by using a series of competing agents that displayed apparent dissociation constants closely similar to corresponding values at a reference biological system. In transfected cells, histamine stimulated, with high potency and large receptor reserve, the accumulation of cAMP. In addition, in the same cells, histamine potently inhibited the release of arachidonic acid induced either by stimulation of constitutive purinergic receptors or by application of a Ca2+ ionophore. This inhibition was independent of either cAMP or Ca2+ levels. The results suggest that a single H2 receptor may be linked not only to adenylyl cyclase activation but also to reduction of phospholipase A2 activity. Because H1 receptors have been reported to stimulate arachidonic acid release, inhibition of this release, an unexpected signaling pathway for H2 receptors, may account for the opposing physiological responses elicited in many tissues by stimulation of these two receptors subtypes.