To date, the majority of characterized extracellular ligand-induced rapid changes in gene expression involve upregulation. Hence, rapid gene repression is either less common or less well studied. To study rapid gene repression during cytokine-initiated differentiation programs, we used the mRNA subtractive hybridization technique of representational difference analysis to isolate repressed genes. Cultures of the myeloid leukemia cell line M1 were induced to terminally differentiate by treatment with interleukin-6 (IL-6). The repressed genes identified in our subtraction products include the genes encoding the growth factor receptor Flt3/Flk2/STK-1 (CD135) and the costimulatory protein CD24 [heat-stable antigen] and the c-myb oncogene. Following 4 h of IL-6 treatment, mRNA levels of these genes are decreased by 45-65% relative to controls and after 8 h by 65-80%. Lipopolysaccharide also triggers the repression of these genes. Protein synthesis inhibitors do not block the IL-6-stimulated repression of c-myb, or c-myc, mRNA, yet they do block the repression of flt3 and CD24 mRNA, demonstrating the existence of both protein synthesis-independent and -dependent mechanisms of cytokine-triggered rapid gene repression during differentiation.