Abstract: In order to investigate the distribution of CDC25B-mRNA in the duodenal mucosa of chicken, the sense and anti-sense digoxigenin (DIG) labeled RNA probe were prepared and utilized. The fragment of CDC25B gene was obtained by RT-PCR through total RNA of chicken embyros. Amplified cDNA fragment was subcloned into pGM-T easy vector, and the plasmid was transformed into E. coli DH5α and screened by “whiteblue plaque selection”. The recombinant plasmid was identified by EcoR Ⅰ restriction enzyme digestion and sequencing, then CDC25B/pGM-T easy vector was linearized with the restriction enzyme of NcoⅠ and SpeⅠ respectively. The sense and anti-sense DIG labeled RNA probe were produced by SP6 and T7 RNA polymerase respectively and transcription in vitro according to the protocol of “DIG RNA Labeling Kit (SP6 /T7)”. The sense and anti-sense RNA probes were prepared successfully. The distribution of CDC25BmRNA in the duodenal mucosa of chicken was examined by in situ hybridization histochemistry( ISHH). There were many labeled cells distributing in the duodenal mucosa of adult chicken. Of these labeled cells, 8160%±963% and 36.21%±8.81% CDC25B-mRNA positive cells were distributed in the basilar part and middle portion of duodenal mucosa of adult chickens respectively. Most of these labeled cells were positive in the regions of the stem cell and proliferation. The positive signals were both in the cytoplasm and cell nucleus. The signals of ISHH were decreased from basilar part to upper in the crypt of lieberkuhn and disappeared in the inferior of villi of small intestine.The labeled cells were both negative in the lamina muscularis mucosae and muscular layer. In conclusion, the sense and anti-sense DIG labeled RNA probes for ISHH of CDC25B were prepared successfully in this experiment, which provided an approach to study further the location of CDC25B-mRNA in chicken. The results of ISHH confirmed the existence of CDC25B-mRNA and athletic proliferation activities in the duodenal mucosa of adult chicken.