The barley (Hordeum vulgare L.) mutants fch2 and clo-f2 comprise an allelic group of 14 Chl b-deficient lines. The genetic map position of fch2 corresponds to the physical map position of the gene encoding chlorophyllide a oxygenase. This enzyme converts chlorophyllide a to chlorophyllide b and it is essential for Chl b biosynthesis. The fch2 and clo-f2 barley lines were shown to be mutated in the gene for chlorophyllide a oxygenase. A five-base insertion was found in fch2 and base deletions in clo-f2.101, clo-f2.105, clo-f2.2800 and clo-f2.3613. In clo-f2.105 and clo-f2.108, nonsense base exchanges were discovered. All of these mutations led to a premature stop of translation and none of the mutants formed Chl b. The mutant clo-f2.2807 was transcript deficient and formed no Chl b. Missense mutations in clo-f2.102 (leading to the amino acid exchange D495N) and clo-f2.103 (G280D) resulted in a total lack of Chl b, whereas in the missense mutants clo-f2.107 (P419L), clo-f2.109 (A94T), clo-f2.122 (C320Y), clo-f2.123 (A94T), clo-f2.133 (A376V) and clo-f2.181 (L373F) intermediate contents of Chl b were determined. The missense mutations affect conserved residues, and their effect on chlorophyllide a oxygenase is discussed. The mutations in clo-f2.102, clo-f2.103, clo-f2.133 and clo-f2.181 may influence electron transfer as illustrated in the active site of a structural model protein. The changes in clo-f2.107, clo-f2.109, clo-f2.122 and clo-f2.123 may lead to Chlb deficiency by interfering with the regulation of chlorophyllide a oxygenase. The correlation of mutations and phenotypes strongly supports that the barley locus fch2 encodes chlorophyllide a oxygenase.