Different experimental conditions have been tested to improve the sequence determination of peptides and proteins by the DABITC (4,4-N,N-dimethylaminoazobenzene-4′-isothiocyanate) method and to facilitate automation in the analysis of the released 4,4-N,N-di-methylaminoazobenzene-4′-thiohydantoin derivatives (DABTHs). Conditions for a complete and rapid separation of all amino acid derivatives have been optimized by using different reversed-phase columns. The stability of the DABTHs in several water-organic solvent mixtures was determined by quantitative analysis and permitted the selection of the appropriate solvents for use in autosamplers. Also the amino acid side-products specific to individual residues which may be observed during thin-layer chromatography of DABTHs can be completely resolved by HPLC and are helpful for a safe assignment of the amino acid residues. The analytical procedures developed have been used to examine the influence of oxygen and detergents on the efficiency of the application of the DABITC manual micromethod on proteins. In the presence of oxygen the recovery of DABTHs is lower in most cases than when the operation is carried out in an inert atmosphere. The presence of a limited amount of detergents does not interfere in the HPLC analysis of DABTHs and, moreover, can increase the efficiency of the sequence analysis of proteins depending on their nature and concentration. In particular, it has been observed that sodium dodecyl sulfate at a concentration of 0.1 % can in some cases produce a threefold increase in the recovery of DABTHs. © 1989.