The expression of substance P in the vocal control and auditory nuclei was investigated by wsing immunohistochemical methods, and the gray density of numerical value was measured with the image processing system in females and males of Carduelis spinus. Then, the distribution and the gray density of substance P were compared between males and females. The results indicate that: 1) Substance P labeled terminal and part cells were distributed in the Area X; 2) Substance P labeled cells were distributed in the nucleus high vocal center (HVc), magnocellular nucleus of the anterior neostriatum (MAN), robust nucleus of the archistriatum (RA) and dorsolateral nucleus of the anterior thalamus (DLM); 3) Substance P labeled terminal and fibers were distributed in the vocal control nuclei such as nucleus dorsalis medialis (DM) and the nucleus hypoglossi, pars tracheosyringealis (nXI-Its), and in the auditory nuclei such as the nucleus ovidalisashell (Ov shell), the shell regions of mesencephalicus lateralis, pars dorsalis (MLd shell) and the nucleus intercollicularis (ICo). The values of gray degree of substance P labeled cells or fibers were significantly higher in males than that in females. The present study indicates that the distribution of substance P exhibits significantly sexual difference in the songbird. The presence of substance P in most auditory and vocal control nuclei suggests that substance P may play an important physiological role in the auditory perception and vocal production.