The Honglian cytoplasmic male sterility (cms-HL) system, a novel type of gametophytic CMS in indica rice, is being used for the large-scale commercial production of hybrid rice in China. However, the genetic basis of fertility restoration (Rf) in cms-HL remains unknown. Previous studies have shown that fertility restoration is controlled by a single locus located on chromosome 10, close to the loci Rf1 and Rf4, which respond to cms-BT and cms-WA, respectively. To determine if the Rf locus for cms-HL is different from these Rf loci and to establish fine-scale genetic and physical maps for map-based cloning of the Rf gene, high-resolution mapping of the Rf gene was carried out using RAPD and microsatellite markers in three BCF1 populations. The results of the genetic linkage analysis indicated that two Rf loci respond to cms-HL, and that these are located in different regions of chromosome 10. One of these loci, Rf5, co-segregates with the SSR marker RM3150, and is flanked by RM1108 and RM5373, which are 0.9 cM and 1.3 cM away, respectively. Another Rf locus, designated as Rf6(t), co-segregates with RM5373, and is flanked by RM6737 and SBD07 at genetic distances of 0.4 cM. The results also demonstrated these loci are distinct from Rf1 and Rf4. A 105-kb BAC clone covering the Rf6(t) locus was obtained from a rice BAC library. The sequence of a 66-kb segment spanning the Rf6(t) locus was determined by a BLASTX search in the genomic sequence database established for the cultivar 93-11.