Diisocyanates, reactive compounds used in plastics industry and potent occupational allergens, readily bind to proteins both in vitro and in vivo , however, the pattern of adducts with individual amino acids has not been investigated systematically. In this study, potential of the proteinogenic amino acid residues for carbamoylation with 2,4-toluenediisocyanate (2,4-TDI) and hexamethylenediisocyanate (HDI) was evaluated. The diisocyanates were incubated in an in vitro system (buffer pH 7.4/dioxane 50:50) with: (a) a series of N α-benzyloxycarbonyl amino acids ( Z-amino acids) and N-acetylcysteine (Ac-Cys), model compounds for non-N-terminal amino acids of the protein chain; (b) dipeptides Val-Phe and Asp-Phe, model compounds for N-termini of globin and albumin, respectively. Reactivity of the compounds tested, evaluated from their depletion during incubation with the diisocyanates (measured by HPLC), was in the order: Ac-Cys=Asp-Phe>Val-Phe= N α- Z-Lys⪢ N α- Z-His for 2,4-TDI, and Ac-Cys>Asp-Phe>Val-Phe= N α- Z-Lys> N α-Z-His>N-Z-Tyr for HDI, however, the adducts with Ac-Cys were unstable. Reactions of other amino acid residues (e.g. Ser, Thr, Met, Trp, Arg, Asn, Gln) with 2,4-TDI and HDI were not observed. Thus, N-terminal amino acids and Lys residues are likely to produce most abundant adducts with diisocyanates in proteins. Further, three amino compounds with increasing p K a values (Val-Phe, Val and N α- Z-Lys) were incubated with 2,4-TDI and N-acetyl- S-[4-(2-amino)tolylcarbamoyl]cysteine, a 2,4-TDI-derived thiocarbamate with carbamoylating activity, in media with 10% and no dioxane, respectively. Here, reactivity of the amino compounds was decreasing in the order: Val-Phe>Val> N α- Z-Lys, which reflects the mechanism of the amine–isocyanate reaction. The experiments also demonstrate the effect of a solvent (organic phase content) on the yield of the carbamoylation reactions.