Cyclic adenosine 5'-diphosphate-ribose (cADPR), a putative Ca²⁺-mobilizing second messenger, has been reported to operate in several mammalian cells. In order to investigate whether cADPR is involved in electrolyte secretion from airway glands, we used a patch clamp technique, the measurement of microsomal Ca²⁺ release, quantification of cellular cADPR, and reversed transcriptase-PCR for CD38 mRNA in human and feline tracheal glands. cADPR (> 6 µM), infused into the cell via the patch pipette, caused ionic currents dependent on cellular Ca²⁺. Infusions of lower concentrations (2 - 4 µM) of cADPR or inositol 1,4,5-trisphosphate (IP₃) alone were without effect on the baseline current, but a combined application of cADPR and IP₃ mimicked the cellular response to low concentrations of acetylcholine (ACh). Microsomes derived from the isolated glands released Ca²⁺ in response to both IP₃ and cADPR. cADPR released Ca²⁺ from microsomes desensitized to IP₃ or those treated with heparin. The mRNA for CD38, an enzyme protein involved in cADPR metabolism, was detected in human tissues including tracheal glands, and the cellular content of cADPR was increased with physiologically relevant concentrations of ACh. We concluded that cADPR, in concert with IP₃, operates in airway gland acinar cells to mobilize Ca²⁺ resulting in Cl^- secretion.