We investigated the cellular regulation of nitric oxide synthase (NOS) activity in isolated acinar cells from rat parotid and human labial salivary glands, using the newly developed fluorescent nitric oxide (NO) indicator, DAF-2. We found that sympathetic stimulation with norepinephrine (NE) caused a strong increase in NO synthesis that was not seen after parasympathetic stimulation with acetylcholine. In rat parotid acinar cells, we furthermore investigated to which extent the NOS activity was dependent on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by simultaneously measuring NO synthesis and [Ca²⁺]_i. It was found that a simple correlation between the rise in [Ca²⁺]_i and the rate of NO production following NE stimulation does not exist, and studies in which [Ca²⁺]_i was elevated by means of the Ca ²⁺ ionophore, ionomycin, further established that even a very large rise in [Ca²⁺]_i did not cause significant NO synthesis. We furthermore found that activating adrenoceptors with NE causes synthesis of cGMP by activating a guanylyl cyclase, and that an enhanced [cGMP] evoked by use of caged cGMP causes Ca²⁺ release from internal stores. Thus, upon sympathetic stimulation, salivary gland acini synthesize NO that, in addition to playing a role in controlling intracellular [Ca²⁺] _i' also might play a role in retrograde signaling processes to the surrounding tissue.