Covalent modification by two-step labeling is illustrated by a blocker-fixer sequence, in which a protein forms a reversible complex with a blocker, which is then converted to an irreversible adduct by the fixer. When the Schiff base-forming enzyme, acetoacetate, decarboxylase (AAD), is used as an in vitro model, the blocker-fixer sequence is exemplified by ethyl acetoacetate (EAA) as the blocker and borohydride as the fixer. When reaction or removal of unbound blocker competes with protein labeling (e.g., with NaBH, as fixer), the quantitative expression for the extent of labeling is @J = 1 - (1 + p), where p is the ratio of the initial blocker concentration, So, to the dissociation constant for the reversible protein-blocker complex. This relationship is tested and demonstrated both for removal of the blocker by chemical reaction and for removal by dialysis. The value of the exponent m is a function of the reaction rate constants. The blocker-fixer sequence EAA-NaBH3CN is shown to be specific for Schiff base-forming sites. Application of this blocker-fixer sequence to the olfactory epithelia of tiger salamanders selectively impairs detection of ketone-containing odorants. When a behavioral assay is used, decrements in responding to cyclopentanone or cyclohexanone are observed at the same time as responding to cyclopentanol, ethyl butyrate, or dimethyl disulfide is unaffected. Dose-response studies show an increase in duration (but not profundity) of selective hyposmia with increasing fixer concentration. On the other hand, the data show a much smaller increase in duration (and no increase in profundity) when the blocker concentration is increased. These results conform to expectations based on the expression for 4 above. They are consistent with the supposition that olfactory ketone receptors with bound ligand behave as do rod cells in the retina in the dark: with a small molecule covalently attached by a Schiff base linkage and with continual secretion of neurotransmitter. Covalent modification of proteins is a widely used tool for examining the action of enzymes.2 Recently, this technique has been extended to the study of receptors in tissue samples. In vivo applications have been less widely probed, for most investigations have not required that the subject survive the chemical treatment. This paper describes the development of a two-step affinity labeling procedure suitable for treating olfactory epithelia of living animals. The two reagents used are a blocker, which binds reversibly to proteins, and a fixer, which converts the reversible complex to an irreversible adduct. Because the experimental subjects remain alive, their sense of smell can be assayed following treatment in order to assess whether the ability to detect specific classes of compounds has been impaired. This paper presents kinetic analyses for two-step labeling schemes, experimental tests using model proteins in solution, and results of application of a two-step labeling procedure to the noses of live tiger salamanders (Am- bystoma tigrinum). The perception of odors is an opportune field for chemical in~estigation.~ There is no complete catalogue of olfactory sensations. Electrophysiological data (much of which has been