Noradrenergic cell specific gene transfer with neuronal nitric oxide synthase (nNOS) can inhibit cardiac sympathetic neurotransmission (unpublished observations). Since neurotransmitter release strictly depends on transmembrane Ca²⁺ influx, we investigated whether selective nNOS gene transfer can modulate calcium handling in isolated cardiac sympathetic neurons. Stellate sympathetic ganglia were dissected from neonatal Sprague Dawley rats and digested using a combination of collagenase and trypsin. Dissociated neurons were purified by panning on a collagen coated dish and plated onto poly-L-lysine/laminin substrate before transduction with an adenoviral vector encoding nNOS driven by a noradrenergic promoter (1). An empty adenoviral vector was used as control for comparing the effect of viral transduction on the neurons. Fura-2 based, ratiometric measurements of intracellular free calcium [Ca²⁺]_i were obtained from cells bathed in HEPES buffered Tyrode solution using a Perkin Elmer UltraView imaging system housed on an inverted Olympus IX70 microscope equipped with a 40×, 1.3 N.A. oil-immersion objective. Ca²⁺ imaging was performed 48 hours post-transduction. Cell depolarization was induced by using 30mM KCl in HEPES Tyrode buffer for 30 seconds. The results showed nNOS gene transferred sympathetic neurons had an 80% lower potassium induced increase in [Ca²⁺]_i compared to neurons transfected with an empty vector. These results demonstrate that noradrenergic neuron-specific gene transfer with nNOS can reduce depolarization-induced increase in [Ca²⁺]_i. The results suggest that nitric oxide (NO) modulation of intracellular Ca²⁺ may be a key step in NO decreasing cardiac sympathetic neurotransmission.