Zinc finger DNA-binding domains can be combined to create new proteins of desired DNA-binding specificity. By shuffling our repertoire of modified zinc finger domains to create randomly generated polydactyl zinc finger proteins with transcriptional regulatory domains, we developed large combinatorial libraries of zinc finger transcription factors (TFZFs). Millions of TFZFs can then be simultaneously screened in mammalian cells. Here, we successfully isolated specific TFZFs that significantly positively and negatively modulate the transcription of the ICAM-1 gene in primary and cancer cells, which are relevant to ICAM-1 biology and tumor development. We show that TFZFs can work in a general and in a cell-type specific manner depending on the regulatory domain and the zinc finger protein. We show that a TFZF that interacts directly with the ICAM-1 promoter at an overlapping NF-kappaB binding enhancer can overcome or synergistically cooperate with NF-kappaB induction of ICAM-1. For this TFZF, rational design was used to optimize the binding of the zinc finger protein to its DNA element and the resulting TFZF demonstrated a direct correlation between increased affinity and efficiency of target gene regulation. Thus, combining library and affinity maturation approaches generated superior TFZFs that may find further applications in therapeutic research and in ICAM-1 biology, and also provided novel mechanistic insights into the biology of transcription factors. Transcription factor libraries provide genome-wide approaches that can be applied towards the development of TFZFs specific for virtually any gene or desired phenotype and may lead to the discovery of new genetic functions and pathways.