Hydroxypyruvate reductase (HPR) is localized in leaf peroxisomes in plants, and it plays an important role in the glycolate pathway of photorespiration. In this laboratory, two highly homologous cDNAs for pumpkin HPR (HPR1 and HPR2) have been obtained, and appear to be produced from the same primary transcript by alternative splicing. Analyses at the mRNA level showed that the amounts of the two HPR mRNAs is changed in response to light, suggesting that light changes the splicing pattern of HPR pre-mRNA from almost equal amounts of two HPR mRNAs to greater production of HPR2 mRNA. From the sequences of the two HPR cDNAs, the HPR1 protein, but not the HPR2 protein, was found to have a targeting sequence into peroxisomes at the carboxy terminus. Analyses of transgenic Arabidopsis thaliana expressing fusion proteins with green fluorescent protein confirmed the different subcellular localizations of the two HPR proteins. These findings indicate the presence of light-regulated alternative splicing of HPR pre-mRNA, which controls the subcellular localizations of two HPR proteins in pumpkin cells.