The globin CACCC boxes are absolutely required for the appropriate regulation of the β-like globin genes. While the β-globin CACCC box binds EKLF/KLF1, a likely adult switching factor, analogous factors, interacting with the γ-globin gene and predicted to regulate the fetal stage of hemoglobin switching, have so far been elusive. By using yeast one hybrid assay, we have isolated four KLFs, KLF1, 2, 4, and 6, that bound the γ-CACCC bait. To establish their role in globin regulation and in the switching of hemoglobins, these factors were compared to four other KLFs already established or putative globin regulators, KLF3, 11, 13 and 16, mainly evaluating their ability to bind and transactivate the ε-, γ- and β-globin gene. γ-CACCC binding at variable intensities was confirmed in band shift assay for all four isolated KLFs, for KLF3 and, faintly, for KLF13. The ε- and β-CACCC were bound by the same factors with similar affinities with the exception of KLF3 and KLF13 that bound stronger to the β- and ε- than to the γ-CACCC box. On the other hand, KLF11 and 16 did not produce any specific complex in band shift assays with anyone of the globin CACCC boxes. More relevant differences were observed among the factors in the transactivation of single and dual luciferase reporters in both K562 and MEL cells. In these assays, most factors presented peculiar modulatory properties and specific promoter tropism. Several factors presented bidirectional activity displaying in the same time the capacity to stimulate and repress different globin promoters. KLF1 and 4 were the strongest stimulators of the β-globin promoter in both cell lines, whereas KLF2 activated the β-promoter only in K562 cells. KLF1 and especially KLF4 consistently repressed ε-globin expression especially in MEL cells. KLF3 behaved always as a general globin repressor in MEL cells, but acted as a weak stimulator of the γ- and ε-promoter in K562 cells. KLF4 was the strongest inhibitor of the ε-globin gene. KLF13 significantly stimulated the γ-promoter in both cell lines, whereas KLF3, 4 and 6 showed statistically significant stimulation only in MEL cells. By RT-PCR analysis we found that KLFs were highly variable in their tissue expression and that KLF1, 3 and 13 had the highest expression in erythroid tissues. Thus the level of tissue expression should ultimately determine which factors are really active in physiological conditions. Taken together our binding and expression studies suggest that several KLFs have the potential to modulate the activity of the globin genes and that the resulting globin expression will depend on the vectorial sum of the relative activities of the factors expressed at any given time of development. Furthermore, as some KLFs, like KLF1 and 4, exert opposite effects on fetal and adult globin genes, their role in hemoglobin switching may be direct and not only dependent on their ability to mediate promoter competition for the LCR.