RALF is a small (5 kDa) and ubiquitous plant peptide signal. It was first isolated from tobacco leaf protein extracts owing to its capacity to alkalinize the extracellular media of cell suspensions. RALFs inhibit root growth and hypocotyl elongation, and a role for RALFs in cell expansion has also been proposed. Arabidopsis has 37 RALF isoforms (AtRALF), but only a small group of nine has high primary structure identity to the original RALF peptide isolated from tobacco. Herein, we report the heterologous production of these nine peptides in Escherichia coli and the evaluation of their activity in five biological assays. All AtRALF peptides produced showed strong alkalinizing activities, with the exception of the pollen-specific isoform AtRALF4. Although it exhibited no inhibitory activity in the root growth and hypocotyl elongation assays, AtRALF4 is a strong inhibitor of pollen germination. Our data demonstrate that the divergence in the tissue specificity and gene expression patterns of the different AtRALFs does not change the fact that their main role seems to be the regulation of cell expansion. Furthermore, different activities in the alkalinization assays upon the addition of two consecutive and saturating doses of the peptides suggest that the peptides are likely being sensed by specific receptors.