The proto-oncogene c-mpl encodes a protein whose sequence shares striking homologies with members of the highly conserved hematopoietin receptor superfamily. This gene had been transduced in a truncated form by the acute leukemogenic murine Myeloproliferative leukemia virus, which exhibits the unique property of inducing factor-independent proliferation and terminal differentiation of a broad spectrum of hematopoietic progenitors. Presently, the ligand and the role of c-mpl in the regulation of normal hematopoiesis are unknown. To show the function of c-mpl, its expression was first examined in human purified hematopoietic cell populations and, then, an antisense strategy was used. By RNA-based polymerase chain reaction, c-mpl transcripts were detected in purified CD34+ cells, megakaryocytes, and platelets. Synthetic unmodified antisense oligodeoxynucleotides were derived from different regions of the c-mpl extracellular domain. On in vitro exposure of CD34+ cells, two antisense oligomers led to a 50% to 70% inhibition of c-mpl mRNA synthesis, whereas their respective sense had no effect. Furthermore, the decrease in c-mpl mRNA correlated with a significant inhibition (range, 54% to 81%) of in vitro megakaryocytic colony formation (CFU-MK), whereas the growth of erythroid (BFU-E) or granulomacrophage (CFU-GM) colonies was unaffected. The data provide first evidences that c-mpl is involved in megakaryocytopoiesis. In addition, the results raise the possibility that this proto-oncogene encodes the receptor for a new cytokine specifically regulating thrombocytopoiesis.