Abstract Retinoic acid (RA) promotes granulocytic differentiation of normal hematopoietic cells and acute promyelocytic leukemia (APL) blasts by transcriptional modulation of myeloid regulatory genes. In this study, we have identified the C/EBP homologous protein (CHOP) as a novel retinoid-responsive gene using a polymerase chain reaction (PCR)-based cDNA subtraction method. All-trans retinoic acid (ATRA) induced a biphasic expression of CHOP mRNA in the NB4 and HL60 AML cell lines. Levels of CHOP expression increased within 1 hour of exposure to ATRA. ATRA expression became nearly absent between 6 and 24 hours, and a second phase of induction occurred after 48 hours. Retinoid-dependent regulation of CHOP expression was also observed in normal human neutrophils but not in peripheral blood mononuclear cells. In addition, retinoid-dependent regulation of CHOP expression was not observed in retinoid-nonresponsive cell lines HL60R and NB4-R2. CHOP expression was regulated at the transcriptional level and was independent of new protein synthesis. CHOP heterodimerized with C/EBPϵ and negatively regulated the myeloid-specific gene lactoferrin. Furthermore, CHOP transcriptionally inhibited C/EBPα- and C/EBPϵ-dependent induction of secondary granule gene expression. RA signaling in granulocytic differentiation involves regulated expression of CHOP and C/EBPϵ in a coordinated fashion. (Blood. 2004;104:3911-3917)