Ag-specific cellular immune responses result in CD4+ T cell activation, which can induce the expression of tissue factor in cells of monocyte/macrophage lineage. This results in initiation of the coagulation protease cascade, with ultimate generation of thrombin. The latter is a potent and pleiotropic mediator of cellular responses and deposition of fibrin. To explore the requirements for extravascular cellular mediation of immune effector pathways, we have searched for a cellular source of the cofactor factor Va. Factor V mRNA was identified in human lymphoid cells by using reverse transcription followed by the polymerase chain reaction (RT-PCR). We confirmed our reverse transcription-polymerase chain reaction results by an independent cloning of factor V cDNA from a T cell cDNA library. The sequence of the factor V cDNA was virtually identical to hepatic factor V mRNA sequence. A limited span of mRNA, encoding part of the connecting region of the factor V protein, was found to contain nucleotide polymorphisms based on six nucleotide substitutions. Northern blot analysis confirmed the presence of a approximately 7-kb factor V mRNA in the Hut-78* human T lymphoma cell line and, at five- to eightfold less abundance, in unstimulated lymphocytes and long term allogeneic stimulated T cells. Immunocytology with factor V mAb identified factor V intracellularly in freshly isolated T lymphocytes but not on the surface of cells. These data provide evidence for factor V transcription and biosynthesis by human lymphocytes. They provide an additional perspective on how lymphocytes may contribute to inflammatory effector functions of cellular immune responses in extravascular sites through provision of cofactors necessary for the coagulation serine protease cascade.