We attempted to engineer a novel long-acting insulin based on the following properties: (i) action as a prodrug to preclude supraphysiological concentrations shortly after injection; (ii) maintenance of low-circulating level of biologically active insulin for prolonged period; and (iii) high solubility in aqueous solution. A spontaneously hydrolyzable prodrug was thus designed and prepared by conjugating insulin through its amino side chains to a 40kDa polyethylene glycol containing sulfhydryl moiety (PEG(40)-SH), employing recently developed hetero-bifunctional spacer 9-hydroxymethyl-7(amino-3-maleimidopropionate)-fluorene-N-hydroxysucinimide (MAL-Fmoc-0Su). A conjugate trapped in the circulatory system and capable of releasing insulin by spontaneous chemical hydrolysis has been created. PEG(40)-Fmoc-insulin is a water-soluble, reactivatable prodrug with low biological activity. Upon incubation at physiological conditions, the covalently linked insulin undergoes spontaneous hydrolysis at a slow rate and in a linear fashion, releasing the nonmodified immunologically and biologically active insulin with a t(1/2) value of 30h. A single subcutaneous administration of PEG(40)-Fmoc-insulin to healthy and diabetic rodents facilitates prolonged glucose-lowering effects 4- to 7-fold greater than similar doses of the native hormone. The beneficial pharmacological features endowed by PEGylation are thus preserved. In contrast, nonreversible, "conventional" pegylation of insulin led to inactivation of the hormone.