Recombinant human IFN α (rhIFN-α) plays an important role in the treatment of hairy cell leukemia (HCL). However, the mechanisms leading to its beneficial effect are not completely clarified, and there is no information on IFN-α gene expression in this disease. Therefore, we investigated the pattern of IFN-α gene expression and protein production in HCL and their potential regulation by rhIFN-α. Blood samples from 10 patients with HCL and 8 healthy donors (HD) were investigated. Expression of IFN-α mRNA was assessed by reverse transcription-PCR analysis in peripheral blood mononuclear cells(PBMCs) under basal conditions and on induction with rhIFN-α and polyionosinic-polycytidylic acid [poly(I·C)]. IFN-αconcentrations in plasma and culture supernatants were measured by immunoassays, and intracellular IFN-α was evaluated by fluorescence-activated cell sorting analysis. Results showed that, in contrast to blood samples from HDs, freshly isolated PBMCs from untreated HCL patients did not express IFN-α mRNA, whereas IFN-α transcripts were found in patients who were under rhIFN-αtherapy. Plasma of untreated patients contained no, or extremely low levels of, IFN-α as compared with plasma of treated patients and HDs. Ex vivo treatment of PBMCs with rhIFN-α or poly(I·C)resulted in a remarkable up-regulation of IFN-α at the mRNA and protein level. In HCL, however, the amounts of IFN-α protein remained less than in HD. Inhibition of IFN-α transcription was found after exposure of PBMCs to serum from untreated patients. Finally, a reduced capacity to produce IFN-α was found within B- cell, T-cell, and monocyte compartments in HCL patients, which could be enhanced by rhIFN-α. The results demonstrate the ability of rhIFN-α to up-regulate the expression of IFN-α gene and protein production and suggest that priming the production of endogenous IFN-α is a critical step in the mechanism of action of rhIFN-α in HCL.