Several thousand plant genes are known to produce multiple transcripts, but the precise function of most of the alternatively encoded proteins is not known. Alternative splicing has been reported for the H-protein subunit of glycine decarboxylase in the genus Flaveria. H-protein has no catalytic activity itself but is a substrate of the three enzymatically active subunits, P-, T- and L-protein. In C(4) species of Flaveria, two H-proteins originate from single genes in an organ-dependent manner. Here, we report on differences between the two alternative H-protein variants with respect to their interaction with the glycine-decarboxylating subunit, P-protein. Steady-state kinetic analyses of the alternative Flaveria H-proteins and artificially produced 'alternative' Arabidopsis H-proteins, using either pea mitochondrial matrix extracts or recombinant cyanobacterial P-protein, consistently demonstrate that the alternative insertion of two alanine residues at the N-terminus of the H-protein elevates the activity of P-protein by 20%in vitro, and could promote glycine decarboxylase activity in vivo.