Zinc finger constitutes one of the most common DNA binding motifs. Although zinc finger proteins consisting of Cys(2)His(2), Cys(3)His, Cys(4), and Cys(6) domains are known in nature, a novel His(4) zinc finger protein has never been observed. Herein, we have created the first artificial His(4)-type zinc finger protein (H(4)Sp1) engineered by Cys --> His mutations of the Cys(2)His(2)type zinc finger transcription factor Sp1. The CD features of the single finger H(4)Sp1f2 and three-finger H(4)Sp1 clearly demonstrate the folding of the mutant His(4) peptides by complexation with Zn(II). The NMR study of Zn(II)-H(4)Sp1f2 reveals that some distortions of the helical region occur due to Zn(II) coordination. The gel mobility shift assay and DNase I footprinting analysis strongly show the binding of Zn(II)-H(4)Sp1 to the GC-box site of duplex DNA. The methylation interference pattern of Zn(II)-H(4)Sp1 binding significantly resembles that of the corresponding C(2)H(2)Sp1 binding. The present artificial peptide H(4)Sp1 is the first example of a zinc finger containing the His4 domain. Of special interest is the fact that the zinc finger domains of H(4)Sp1 are folded (although not identical to the native structure) and bind DNA similar to wild-type C(2)H(2)Sp1.