Krüppel-like factors (KLFs) have been systematically screened as potential candidates to regulate human gamma-globin gene expression through its CACCC element. Initially, 21 human proteins that have close sequence similarity to EKLF/KLF1, a known regulator of the human beta-globin gene, were identified. The phylogenetic relationship of these 22 KLF/Sp1 proteins was determined. KLF2/LKLF, KLF3/BKLF, KLF4/GKLF, KLF5/IKLF, KLF8/BKLF3, KLF11/FKLF, KLF12/AP-2rep and KLF13/FKLF2 were chosen for functional screening. Semi-quantitative RT-PCR demonstrated that all eight of these candidates are present in human erythroid cell lines, and that the expression of the KLF2, 4, 5 and 12 mRNAs changed significantly upon erythroid differentiation. Each of the eight KLF mRNAs is expressed in mouse erythroid tissues, throughout development. UV cross-linking assays suggest that multiple erythroid proteins from human cell lines and chicken primary cells interact with the gamma-globin CACCC element. In co-transfection assays in K562 cells, it was demonstrated that KLF2, 5 and 13 positively regulate, and KLF8 negatively regulates, the gamma-globin gene through the CACCC promoter element. The data collectively suggest that multiple KLFs may participate in the regulation of gamma-globin gene expression and that KLF2, 5, 8 and 13 are prime candidates for further study.