Sequence-based gene isolation has been a practical approach for plant resistance gene cloning. In this study, RS13, a cloned rice sequence with the NBS (nucleotide-binding site) domain of resistance genes, was used as a probe to screen a bacterial artificial chromosome (BAC) library of rice variety IR64,and four positive clones were obtained. Of them the clone 14E19 covered the other three clones and was sequenced through a shotgun approach. The whole sequence of the insert fragment of 14E19 was assembled into approximately 73 kb in length. Genes on the whole assembled sequence were predicted,and four genes encoding NBS and LRR (leucine-rich repeat) domains were found, named as NL-A, B, C and D respectively. For further analysis, another longer BAC clone,106P13, covering 14E19 on the same chromosome position was identified from a BAC library of IRBB56 which had the same genome background with IR64. Ten NL-homologous copies were discovered on the sequence of the BAC clone 106P13, and four copies were identical with those on 14E19. The similar homologous sequences were also found in the genomic sequences of Nipponbare,93-11 and Guangluai4. However, NL sequences were less homologous with the known NBS-LRR resistance genes. This result indicated that NL was a new NBS-LRR gene family and was composed of ten members at least. RT-PCR and cDNA screening displayed that NL-B expressed in a bacterial blight-resistant rice variety IRBB4, indicating the gene was possibly involved in resistance reactions.