Winged helix factors are important regulators of embryonal development and tissue differentiation. They are also involved in translocations found in acute leukemias and solid tumors. We have detected transcripts from five known and four novel winged helix genes in leukemia cell lines and CD34(+) blood progenitor cells by reverse trancription-polymerase chain reaction with degenerate primers on the highly conserved DNA binding domain. The genomic clones coding for two new winged helix proteins, FOXD4a and FOXD4b were isolated by high-stringency hybridization of a human phage library. FOXD4a and FOXD4b are encoded by a 1319 and 1250 bp single exon coding for a winged helix DNA binding domain, an amino-terminal acidic region and a carboxy-terminal proline- and alanine-rich region which correspond to putative transcriptional regulatory motifs. TATA box, CCAAT box, and transcription factor binding motifs have been identified in the 5' region of the genes. In addition, foxD4a and foxD4b cDNA has been isolated from NB-4 mRNA. The fox genes are transcribed in a tissue-restricted pattern in adult and fetal human tissues. FoxD4a and foxD4b mRNA was expressed in the leukemia cell lines KG-1, Kasumi, NB-4, HL-60, U937, THP-1, HEL, U266, Jurkat, and Raji. It has already been shown that winged helix factors are also involved in carcinogenesis. Based upon these studies, our results suggest that FOXD4a and FOXD4b may play a role in leukemogenesis.