Photoperiod-sensitive genic male-sterile (PSGMS) rice, in which pollen fertility is regulated by day-length, originally arose as a natural mutant in the rice cultivar Nongken 58 (Oryza sativa ssp. japonica). Previous studies identified pms3 on chromosome 12 as the locus of the original PSGMS mutation. In this study we have assigned the pms3 locus to a 28.4-kb DNA fragment by genetic and physical mapping. A cross between Nongken 58S (PSGMS line) and DH80 was used to produce an F2 population of about 7000 plants, from which 892 highly sterile individuals were obtained for recombination analysis. By analyzing recombination events in the sterile individuals using a total of 157 RFLP probes from a BAC contig covering the pms3 region, the pms3 locus was localized to a sub-region of less than 1.7 cM. Further analysis of recombination events using 49 additional probes isolated from this sub-region identified markers flanking the pms3 region on each side; these markers are only 28.4-kb apart. Sequence analysis of this fragment predicted the presence of five ORFs, found high homology with two ESTs in public databases, and detected three SNPs between the mutant and the wild-type parents, which may be helpful for identifying a candidate gene for pms3.