The forkhead gene (FKH) regulates morphogenesis in Drosophila. It is the prototype of a new family of transcriptional activators. Partially degenerate oligonucleotides to two conserved amino acid sequences of this family were used to prime a polymerase chain reaction (PCR) amplification of HEL cell cDNA. Two unique clones, designated H3 and H8, were isolated that contained homologies to FKH. A third novel clone, 5-3, was isolated by low stringency screening of a chronic myelogenous leukemia cDNA library using H8 as a probe. H3 and 5-3 are preferentially expressed in restricted hematopoietic lineages, while the expression of H8 was ubiquitous. Southern analysis showed that FKH 5-3 is conserved through yeast, which is rare among tissue-specific transcription factors. The H3 and 5-3 clones provide evidence that FKH family members are present in a tissue-restricted manner in humans.