Granule-bound starch synthase (GBSS), a product of the waxy gene in rice (Oryza sativa L.), is necessary for the synthesis of amylose in the endosperm. In an extended pedigree of 89 rice cultivars, we have previously shown that all cultivars with more than 18% amylose had the sequence AGGTATA at the leader intron 5' splice site, while all cultivars with a lower proportion of amylose had the sequence AGTTATA. This single-nucleotide polymorphism reduces the efficiency of GBSS pre-mRNA processing. It also results in alternate splicing at multiple sites, some of which have non-consensus sequences. Here we demonstrate that this same G-to-T polymorphism is also associated with differential sensitivity to temperature during the period of grain development. Cultivars with the sequence AGTTATA have a substantial increase in accumulation of mature GBSS transcripts at 18 degrees C compared to 25 or 32 degrees C. The selection of leader intron 5' splice sites is also affected by temperature in these cultivars. A 5' splice site -93 upstream from that used in high-amylose varieties predominates at 18 degrees C. At higher temperatures there is increased utilization of a 5' splice site at -I and a non-consensus site at +1. Potential implications of differential 5' splice site selection and associated differences in 3' splice site selection on transcript stability and translational efficiency are discussed.