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.