A cDNA clone, named gut-enriched Krüppel-like factor (GKLF), was isolated from an NIH 3T3 library using a probe encoding the zinc finger region of the immediate-early transcription factor zif/268. The deduced GKLF amino acid sequence contains three tandem zinc fingers that are related to members of the Krüppel family of transcription factors. By indirect immunofluorescence, GKLF is localized to the cell nucleus. In cultured fibroblasts, GKLF mRNA is found in high levels in growth-arrested cells and is nearly undetectable in cells that are in the exponential phase of proliferation. The growth-arresting nature of GKLF is demonstrated by an inhibition of DNA synthesis in cells transfected with a GKLF-expressing plasmid construct. In the mouse, GKLF mRNA is present in select tissues and is most abundant in the colon, followed by the testis, lung, and small intestine. In situ hybridization experiments indicate that GKLF mRNA is enriched in epithelial cells located in the middle to upper crypt region of the colonic mucosa. Taken together, these results suggest that GKLF is potentially a negative regulator of cell growth in tissues such as the gut mucosa, where cell proliferation is intimately coupled to growth arrest and differentiation.