Twenty-one human immunodeficiency virus (HIV)-positive patients, including 11 acquired immunodeficiency syndrome (AIDS)-free patients with immune thrombocytopenic purpura (ITP), were studied to determine whether the megakaryocytic/platelet lineage was infected by HIV. Because purification of platelets did not reach a level sufficient for unequivocal results by the polymerase chain reaction, in situ hybridization was thus performed. Purified marrow megakaryocytes (MK) from 10 HIV-infected ITP patients were studied using a 35S HIV riboprobe, antisense of an HIV ENV sequence. HIV transcripts were clearly detected in MK from five of these 10 patients, although heterogeneity among MK was observed. In three of these five cases, small amounts of HIV glycoproteins were detected in MK by means of immunofluorescence. In addition anti-HIV antibodies could be eluted from platelets of all patients. In contrast, HIV transcripts were not detected in MK derived from colony-forming units-MK (CFU-MK) cultured in suspension, suggesting either that MK are infected by HIV during terminal differentiation or that HIV-infected CFU-MK are unable to differentiate in vitro. In conclusion, this study suggests that HIV infection of MK may be implicated in the pathogenesis of thrombocytopenia of HIV-positive patients.