Using Western blot and fluorescent immunocytochemistry, NOS III (or ecNOS) and NOS II (or iNOS), but no NOS I (or ncNOS), were identified in preparations of human platelets. Reverse-transcription polymerase chain reactions (RT-PCR) demonstrated NOS III mRNA, but no NOS II mRNA (which is short-lived) and no NOS I mRNA in platelets. Immunofluorescent staining of human bone marrow smears showed the presence of NOS III, but not NOS I in megakaryocytes. A subpopulation of megakaryocytes also expressed NOS II. In preparations of human neutrophils, immunocytochemistry demonstrated NOS I in all cells, whereas no NOS III was detected. The few NOS II positive cells were characterized as contaminating eosinophils. Similarly, in RT-PCR, transcripts for NOS I and NOS II, but not for NOS III, were identified. Thus, the constitutive NOS isoform in megakaryocytes and platelets is NOS III, whereas neutrophils express NOS I. Some megakaryocytes and eosinophils also express NOS II.