Increasing leaf photosynthesis offers a possible way to improve yield potential in rice (Oryza sativa L.). Carbon isotope discrimination (Delta(13)C) has potential as an indirect selection criterion. In this study, we searched for quantitative trait loci (QTLs) controlling Delta(13)C, and assessed their association with leaf photosynthesis. Substitution mapping by using chromosome segment substitution lines (CSSLs), that carry segments from the indica cultivar Kasalath in the genetic background of the japonica cultivar Koshihikari, identified genomic regions affecting Delta(13)C on chromosomes (Chr.) 2, 3, 6, 7, and 12. One of the CSSLs, SL208, in which most regions on Chr. 3 were substituted with Kasalath segments, showed higher leaf stomatal conductance for CO(2) (g (s)) and Delta(13)C than Koshihikari during the vegetative stage although leaf photosynthetic rate did not differ between them. These results suggest an association between Delta(13)C and g (s). To test this association, we performed a QTL analysis for Delta(13)C at vegetative and heading stages in an F(2) population derived from a cross between SL208 and Koshihikari. The results confirmed a QTL controlling Delta(13)C on the long arm of Chr. 3. By using a near-isogenic line specific to Hd6, we ruled out the possibility that variation in Delta(13)C was generated through the pleiotropic effect of heading date.