A recent paper reports on the occurrence of alternative splicing of H-protein pre-mRNA in the C4 species, Flaveria trinervia, that is organ-specifically regulated. The analysis of 11 other species of the genus, F. cronquistii and F. pringlei (C3), F. anomala, F. chloraefolia, F. floridana, F. linearis and F. pubescens (C3-C4 intermediate), F. brownii (C4-like), F. palmeri, F. bidentis and F. australasica (C4), revealed that this post-transcriptional effect is not specific for F. trinervia. It occurs in all the examined C4 species of the genus Flaveria except the less advanced C4-like species, F. brownii. Both the position and the direct effect of alternative splicing, the addition of two alanine residues near to the N-terminus of the derived mature H-protein, are invariant. A quantification of the relative amounts of both transcripts revealed that, as in F. trinervia, the alternative mRNA strongly dominates in leaves. In sharp contrast, none of the C3, C3-C4 intermediate, or C4-like species showed alternative splicing. By Western analysis both H-isoproteins have been detected in F. trinervia and their ratio approximately corresponds to the measured transcript levels. It is concluded that alternative splicing leads to the synthesis of two different H-proteins of the glycine cleavage system in all advanced Flaveria C4 species.