The autonomic receptors of the rat sublingual gland were characterized by radioligand binding and by specific functional responses involving the release of K⁺ and the generation of cyclic AMP in vitro. Both muscarinic cholinergic and alpha₂-adrenergic receptors were present in moderately high density in the sublingual gland, as judged by the binding of the specific radioligands ³H-QNB and ³H-clonidine (B _max in pmol/g tissue=18.4±2.4 and 9.9±1.3, respectively). By contrast, although alpha₁ and beta-adrenergic receptors were detected, they were not present in large numbers. The B _max (pmol/g tissue) for the binding of ³H-prazosin and ³H-DHA were, respectively, 3.2±0.6 and 3.6±0.4. Stimulation of muscarinic cholinergic receptors with carbamylcholine (2×10^–5 M) caused a net release of K⁺ from sublingual slices incubated in an enriched, oxygenated Krebs Ringer bicarbonate medium of 35±7% after 10 min of incubation. Norepinephrine, phenylephrine, clonidine and isoproterenol did not induced K⁺ release from the slice preparation, but actually reduced the basal or unstimulated release of K⁺. As in the submandibular and parotid glands, the release of K⁺ from sublingual slices had two components, a passive efflux and an active uptake which depended on the activation of an ouabain-sensitive Na⁺, K⁺ ATPase. The release of K⁺ was also dependent on the presence of Ca²⁺ (2.7 mM) in the incubation medium. Stimulation of beta-adrenergic receptors with isoproterenol (10^–5 M) caused a 9-fold increase in the content of glandular cyclic AMP (from 4.0±0.6 to 36.0±2 pmol/mg/10 min). A similar increase was observed with norepinephrine (10^–4 M) in the presence of phentolamine (10^–4 M). Clonidine at concentrations of 10^–5 and 10^–4 M reduced the cyclic AMP content to below basal levels. The rat sublingual gland has functional cholinergic receptors and can release K⁺ in vitro in the presence of cholinergic agents. In contrast to the other major salivary glands, it has a low density of alpha₁-adrenergic receptors and fails to release K⁺ upon stimulation with alpha₁-agonists. The functional significance of a low density of beta-adrenergic receptors is still unclear, although there is a definite glandular cyclic AMP response upon stimulation with isoproterenol. Abundant alpha₂-adrenergic receptors in the sublingual gland are apparently negatively coupled to adenylate cyclase.