The IF1 protein is one of the factors controlling translation initiation in bacteria. This protein is encoded by the infA gene, which, in several higher plants, is located in the plastome. Cytoplasmic Line 2 (CL2), an alboviridis barley mutant, was the first to be proposed as an infA gene mutation (T 157 C) in higher plants. This mutant was isolated from a chloroplast mutator genotype (cpm/cpm) and was made genetically stable by backcrosses with a wild-type nuclear genotype. In the present work, genetically stable CL2 plants were backcrossed as females by cpm/cpm plants in order to regain the mutator activity. Interestingly, a seedling carrying a first leaf blade with a darker green stripe on a typical CL2-mutant background was observed in the F(4) generation. The T 157 C transition was confirmed in tissues from the CL2 background, whereas a second transition (A 178 G) was also found in the darker stripe. Two clearly different levels of CL2 syndrome were observed in the seedlings of the F(5) and F(6) progenies. Those of the greener group carried both transitions. These results suggest a compensatory effect of the second mutation and support the involvement of the infA plastid gene in CL2 syndrome, confirming CL2 as the first mutant of this gene reported in higher plants.