Previous studies have suggested the presence of nitric oxide synthase (NOS) enzyme in human platelets. We herein provide definitive evidence for the presence of both endothelial constitutive NOS (ecNOS) and inducible NOS (iNOS) isoforms and their mRNA in human platelets. Total RNA was isolated from human platelets, and reverse-transcription polymerase chain reaction (RT-PCR) demonstrated the expression of the ecNOS and iNOS isoforms in platelets. High-stringency Southern analysis confirmed the molecular authenticity of the RT-PCR products for each NOS isoform. Western analysis with mouse monoclonal antibody against human ecNOS consistently demonstrated a band with a molecular weight of 140-150 kDa. Western analysis with mouse monoclonal antibody against rat macrophage iNOS showed a single 200-kDa band in both resting and lipopolysaccharide (LPS)-plus interferon-gamma (IFN-gamma)-stimulated platelets. Immunoprecipitation further confirmed the presence of the 200-kDa iNOS band. Expression of iNOS protein, measured with densitometry, was increased in LPS- and IFN-gamma-stimulated platelets (p < 0.01 vs. resting platelets). Thus, human platelets possess both ecNOS and iNOS isoforms and their mRNA, and iNOS exhibits molecular weight and kinetic characteristics distinct from those of ecNOS.