Bovine adrenal chromaffin cells share many characteristics with neurons, and are often used as a simple model system to study ion channels and neurotransmitter release. We infected bovine adrenal chromaffin cells with a replication deficient adenovirus that induces expression of the common reporters beta-Galactosidase and Green Fluorescent Protein via a bicistronic sequence. In perforated patch recordings performed 48 h post-infection, peak calcium currents were reduced 32%, primarily due to loss of omega-conotoxin-GVIA-sensitive current. In contrast, sodium currents were increased 17%. Exocytosis, detected as an increase in membrane capacitance immediately after a single step depolarization, was reduced in proportion to the decrease in calcium influx. However, capacitance continued to increase for seconds following the depolarization. The amplitude of this post-stimulus drift, or asynchronous exocytosis, was ~ 3 times that which occurred in a small fraction of control cells. Exocytosis evoked by repetitive stimulation with a train of brief depolarizations was increased 50%. Intracellular calcium levels measured during and following stimulation were lower, not higher, in adenovirus infected cells. Electroporated cells showed reduced calcium currents but no enhancement of exocytosis. Cells infected with UV-irradiated virus showed reduced calcium currents and enhancement of exocytosis, but the changes were smaller than those caused by the intact virus. Our results are consistent with the idea that adenovirus capsid and adenoviral DNA contribute to a Ca(2+) influx- and [Ca(2+)]i-independent enhancement of exocytosis in bovine chromaffin cells.