Rice heme oxygenase 2 (OsHO2) mutants are chlorophyll deficient with distinct tetrapyrrole metabolite and transcript profiles, suggesting a potential regulatory role of the stromal-localized OsHO2 in tetrapyrrole biosynthesis. In plants, heme oxygenases (HOs) are classified into the subfamilies HO1 and HO2. HO1 are highly conserved plastid enzymes required for synthesizing the chromophore in phytochromes which mediate a number of light-regulated responses. However, the physiological and biochemical functions of HO2, which are distantly related to HO1, are not well understood, especially in crop plants. From a population of (60)Coγ-irradiated rice mutants, we identified the ylc2 (young leaf chlorosis 2) mutant which displays a chlorosis phenotype in seedlings with substantially reduced chlorophyll content. Normal leaf pigmentation is gradually restored in older plants while newly emerged leaves remain yellow. Transmission electron microscopy further revealed defective chloroplast structures in the ylc2 seedlings. Map-based cloning located the OsYLC2 gene on chromosome 3 and it encodes the OsHO2 protein. The gene identification was confirmed by complementation and T-DNA mutant analyses. Subcellular localization and chloroplast fractionation experiments indicated that OsHO2 resides in the stroma. However, recombinant enzyme assay demonstrated that OsHO2 is not a functional HO enzyme. Analysis of tetrapyrrole metabolites revealed the reduced levels of most chlorophyll and phytochromobilin precursors in the ylc2 mutant. On the other hand, elevated accumulation of 5-aminolevulinic acid and Mg-protoporphyrin IX was observed. These unique metabolite changes are accompanied by consistent changes in the expression levels of the corresponding tetrapyrrole biosynthesis genes. Taken together, our work suggests that OsHO2 has a potential regulatory role for tetrapyrrole biosynthesis in rice.