In order to investigate the effects of long-term application of nitrogen fertilizer on soil denitrifying communities, the diversities of nir genes (nirK and nirS) were studied using molecular approaches in the long-term paddy field experiment (started in 1990) located in Taoyuan. Analysis of clone sequences indicated that the nirK fragments from paddy soil showed close similarity (90.7%) to the nirK sequences registered in GenBank database, but were not related to any known strain. Whereas, most of the airS clones showed low similarity (74.7%) to the nirS gene fragments registered in GenBank. The Chao1 estimates showed that the diversity of nirK gene 13) OTUs] than in N treatment [(49 +/- 9) OTUs], but the difference was not significant. However, application of nitrogen fertilizer resulted in significant difference of nirS-community compared to CK. Nitrogen fertilizer had obvious effect on tbe community structure of nirK-denitrifiers (p < 0.022), but the nirS-containing community was not affected. Based on phylogenetic analysis, nirK clones grouped into three clusters with aggregations of some OTUs cloned from N treatment. Although nirS clones grouped into four clusters, the majority of the clones were attributed in one cluster. The results suggested that application of nitrogen fertilizer had a greater influence on the diversity of nirS-containing bacterial community than that of the nirK. However, the community structure of nirK-containing denitrifiers was more sensitive to nitrogen fertilization than that of the mrS.