Abstract: The fragment of SP gene was obtained by RT-PCR through total RNA of chicken brains. The amplified cDNA fragments were subcloned into pGM-T vector, and the plasmids were transformed into E. coli TOP10 and chosen by “white-blue plaque selection”. The recombinant plasmids were identified by sequencing. SP/pGM-T vectors were then linearized with the restriction enzyme of Nco Ⅰ and Sal Ⅰ, respectively. The sense and anti-sense DIG labeled RNA probes were producted by using SP6 and T7 RNA polymerase, respectively and in vitro transcription according to the protocol of “DIG RNA Labeling Kit (SP6/T7)”. Certificated by dot blot hybridization, the sense and antisense RNA probes were prepared successfully. The distribution of SP-mRNA in chicken INR was examined by using in situ hybridization histochemistry (ISHH). Most positive cells had oval and other polygonal shapes, and the labeled neurons distributed in ganglion in layers or small groups and in nerve trunk between ganglions sporadically. The proportion of positive cells in total neurons was 82.98% in the juxta-jejunoileum portion and 98.01% in the juxta-rectal portion of INR. In conclusion, the sense and anti-sense DIG labeled RNA probes for ISHH of SP were prepared successfully in this experiment which provided an approach for the further study of the location of SP-mRNA in nerve tissue. The results of ISHH suggest the existence of the SP-mRNA in most neurons of INR.