Polypeptide drugs are generally short-lived species in circulation. In this study, we have covalently linked seven moieties of 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) to the amino groups of human interferon-α2. The derivative thus obtained (FMS₇–IFN-α2) has ≈4% the biological potency and 33 ± 4% the receptor binding capacity of the native cytokine. Upon incubation, FMS₇–IFN-α2 undergoes time-dependent spontaneous hydrolysis, generating active interferon with t_1/2 values of 24 ± 2 h at pH 8.5 and 98 ± 10 h at pH 7.4. When native IFN-α2 is intravenously administered to mice, circulating antiviral activity is maintained for a short duration and then declines with t_1/2 = 4 ± 0.5 h, reaching undetectable values at ≈18 h after administration. With intravenously administered FMS₇–IFN-α2, there is a lag period of 2 h, followed by a progressive elevation in circulating antiviral-active protein, which peaked at 20 h and declined with t_1/2 = 35 ± 4 h. FMS₇–IFN-α2 is resistant to α-chymotrypsin digest and to proteolytic inactivation by human serum proteases in vitro. We have thus introduced here an inactive IFN-α2 derivative, which is resistant to in situ inactivation and has the capability of slowly reverting to the native active protein at physiological conditions in vivo and in vitro. Having these attributes, FMS₇–IFN-α2 maintains prolonged circulating antiviral activity in mice, exceeding 7–8 times the activity of intravenously administered native cytokine.