Cytoplasmic line 2 (CL2) is a chlorophyll mutant that was selected from a plastid mutator genotype in barley. The dynamics of greening and plastid development of CL2 first-leaf blade contrasts with that of monocots. Previous characterizations of CL2 suggested that this mutant has a delay of plastid gene translation during embryogenesis. We hypothesize that CL2 is a mutant in the infA gene, which encodes translation initiation factor 1 (IF1). Wild-type barley infA gene differs in some nucleotides from that in wheat, but the corresponding IF1 proteins are identical. However, infA from CL2 carries a point mutation, which leads to an amino acid change in IF1 residue 52. One CL2-like seedling selected from a new mutator pool also carries a point mutation in infA gene, this time leading to a change of the universally conserved amino acid residue 32. Both point mutations were T --> C substitutions. We sequenced the complementary DNA of the infA transcripts from the wild type and CL2 and found that the mutation was conserved at the mRNA level. Results strongly suggest that CL2 and CL2-like are infA gene mutants, this being the first time that a mutant phenotype is attributed to infA gene in a higher plant.