Semi-dominant Oil yellow1 (Oy1) mutants of maize (Zea mays) are deficient in the conversion of protoporphyrin IX to magnesium protoporphyrin IX, the first committed step of chlorophyll biosynthesis. Using a candidate gene approach, a cDNA clone was isolated that was predicted to encode the I subunit of magnesium chelatase (ZmCHLI) and mapped to the same genetic interval as Oy1. Allelic variation was identified at ZmCHLI between wild-type plants and plants carrying semi-dominant alleles of Oy1. These differences revealed putative amino acid substitutions that could account for the alterations in protein function. Candidate lesions were tested by introduction of homologous changes into the Synechocystis magnesium chelatase I gene (SschlI) and characterization of the activity of mutant protein variants in an in vitro enzyme activity assay. The results of these analyses suggest that SsChlI protein variants containing the substitutions identified in the dominant Oy1 maize alleles lack activity necessary for magnesium chelation and confer a semi-dominant phenotype via competitive inhibition of wild-type SsChlI.