During thylakoid membrane biogenesis, chlorophyll (Chl) biosynthesis and the accumulation of Chl-binding proteins are tightly linked, light-regulated processes. We have investigated the consequences faced by mutant plants with defects in Chl biosynthesis by studying a series of five homeologous allelic chlorina mutants in wheat (Triticum) and one phenotypically related barley (Hordeum vulgare) mutant that express the same pleiotropic mutant phenotype but to different extents. These mutants accumulate Chl at different rates, with the most severely affected plants having the slowest rate of Chl accumulation. Analysis of precursor pools in the Chl synthesis pathway indicates they have a partial block in Chl synthesis and accumulate protoporphyrin IX (Proto), the last porphyrin compound common to both heme and Chl synthesis. The affected plants with the most severe phenotypes accumulate the most Proto. Chloroplasts isolated from these mutants exhibit a lower activity of the enzyme Mg-chelatase, which catalyzes the first committed step in Chl synthesis. The most severely affected plants exhibit the greatest reduction in Mg-chelatase activity. Heme levels and protoporphyrinogen oxidase activity were the same for mutant and wild-type plants. We suggest that a block in Mg-chelatase activity in these mutants could account for the other traits of their pleiotropic phenotype previously described in the literature.