Objective To investigate the expression of histamine in the cardiac sympathetic fibers from the guinea pig superior cervical ganglion and its coexistence with norepinephrine so as to provide morphological evidence for histamine as a cardiac sympathetic neurotransmitter. Methods Biotinylated dextranamine (BDA) anterograde tracing and immunofluorescence histochemical staining for histamine/norepinephrine were applied. Results After injection of BDA into the superior cervical ganglion, BDA labeled sympathetic fibers in the left and right atria and ventricle were observed. Meanwhile, the tracing fibers proved histamine-like immunoreactive or both histamine and norepinephrine-like immunoreactive. Conclusion Histamine is expressed in the cardiac sympathetic fibers from the guinea pig superior cervical ganglion and coexisted with norepinephrine.