Several neuropeptides have been demonstrated to coexist with classical neurotransmitters in the central and peripheral nervous systems and have been proposed as neurotransmitter or neuromodulator candidates. In this report, we investigated the relationship between the external calcium concentration and the electrically induced overflow of noradrenaline (NA) and neuropeptide Y (NPY) in dog perfused spleen. Perfusion solutions with calcium concentrations ranging from 0 to 10 mM were applied. The splenic nerves were electrically stimulated at 16 Hz. For NA analysis high-pressure liquid chromatography with electrochemical detection was used. NPY was determined by radioimmunoassay. The perfusion pressure and the overflow of NA and NPY increased in a calcium concentration dependent manner. The calcium concentration dependency of the overflow of NA and NPY was comparable, indicating a co-release of the two substances. The molar ratio of NA/NPY remained unchanged over the calcium concentration range applied and the half maximal saturation values for release of NA (5.55 mM) and NPY (6.66 mM) were similar. These results indicate that the preferential release of NPY at high frequency stimulation as previously shown in the pig spleen, if present in the dog, is not the result of a difference in calcium dependency of the evoked release of NA and NPY.