Interleukin-3 (IL-3) is a potent stimulator of megakaryocyte proliferation, and autocrine production of IL-3 by megakaryocytic leukemia cell lines and bone marrow-derived megakaryocytes has recently been demonstrated. To characterize the transcriptional regulation of IL-3 in megakaryocytes, we transiently transfected IL-3 promoter CAT constructs that contain variable amounts of 5' flanking sequences into the human CMK and CMK-6 megakaryocytic cell lines and identified two positive acting transcriptional regulatory regions, one located between bp -315 and -284, which contains consensus AP-1 and ets binding sites, and a second located between bp -173 and -61. DNase I footprinting assays using CMK or CMK-6 nuclear extracts demonstrate DNA-protein interactions in the identical region protected by T cell or natural killer cell nuclear extracts (between bp -165 to -128), and electrophoretic mobility shift assays demonstrate the binding of proteins to three distinct portions of this region. To characterize the transcription factors in megakaryocytic cells that could bind to these two regulatory regions, we performed Northern blot analyses, which showed the presence of ets-1, elf-1 (which is thought to be restricted to T cells), NF-IL3A and AML1 mRNAs, as well as c-fos, jun B, and jun D, but not c-jun mRNA. These studies show that the transcriptional regulation of IL-3 expression in megakaryocytic leukemia cell lines is similar, but not identical to normal human T cells.