Some patients treated with interferon (IFN) have developed antibodies (ABs) to that IFN. We examined the incidence of such ABs in hairy cell leukemia (HCL) patients treated with IFN-alpha 2a and IFN-alpha 2b. In the initial enzyme-linked immunosorbent assay (ELISA) assays, the serum samples were tested against their corresponding IFNs. Of 73 evaluable patients treated with IFN-alpha 2b, 6 patients, who tested negative prior to treatment, were positive by ELISA following treatment with IFN. Two of the samples tested positive in the neutralization assay. Regarding the samples from patients treated with IFN-alpha 2a, 2 of the 44 patients were ELISA positive following IFN treatment (while being negative prior to treatment) and 2 others became positive in the neutralization assay following IFN treatment. Five serum samples with neutralizing activity were tested for their ability to bind to each of eight natural IFN-alpha species derived from human lymphoblastoid cells induced with Sendai virus; a different cross-reactivity profile was seen for each sample. In conclusion, a low incidence of neutralizing and nonneutralizing ABs against IFN-alpha 2 was observed in patients with HCL treated with recombinant DNA-derived IFN-alpha 2a or IFN-alpha 2b. Finally, ABs against these IFNs cross-react by ELISA with several naturally occurring IFN-alpha s.