Abstract: A FIA system equipped with a spectrophotometric detector and a computerized data system was used. The carrier stream was 0.02 M carbonate buffer of pH 10.5 or 11 containing phenolphthalein or 4-nitrophenolate, respectively, as absorbing guest micromolecule. The injected solution contained β- or α-cyclodextrin, respectively, in the same buffer. Absorbance measurements of the transient systems formed during complexation were used to determine the formation constants. Competitive complexation studies were also carried out with phenolphthalein as probe to determine the formation constants of the β-cyclodextrin complexes of 2-(cyclohexylamino)ethanesulfonic acid, 3-(cyclohexylamino)propanesulfonic acid and naproxen; the carrier contained the probe and the micromolecule and the injected solution comprised the same solution with β-cyclodextrin added. Results showed reasonable agreement with literature values. An automated gradient flow injection (FI) spectrophotometric technique for the determination of formation constants of complexes of micromolecules with cyclodextrins (CDs) is described. Only two injections are required for the determination of the CD-micromolecule formation constant. The concentration gradient is calibrated by injecting a dye solution in a buffer carrier. A mixed CD-micromolecule solution is injected afterward into a flowing stream of buffered micromolecule solution. By the use of a mixing chamber, a great number of transient 'mixed solutions' are created and their absorbance readings are used for the determination of the CD-micromolecule formation constant. Formation constants for phenolphthalein with b-CD (K = 2.4(0.2) 104 M-1 in carbonate buffer pH 10.5, J = 23.5°C) and p-nitrophenolate with a-CD (K = 2.0(0.2) 103 M-1 in carbonate buffer pH 11.0, J = 24.5°C) were determined on the basis of spectral changes upon complexation by CDs. Formation constants of the complexes of the buffer species CHES (K = 4.9(0.2) 102 M-1 in NaOH pH 10.5, J = 27.0°C) and CAPS (K = 4.8(0.5) 102 M-1 in NaOH pH 11.5, J = 25.0°C) and the drug naproxen (K = 6.3(0.5) 102 M-1 in 0.020 M Na2CO3 pH 10.5, J = 25.0°C) with b-CD were also determined by adapting the competitive spectrophotometric indicator approach to the FI gradient technique. Formation constants determined with the proposed method are in reasonable agreement with literature values. This technique utilizes the total information contained in FI peaks, requires minimal amounts of CD, and provides accurate and fast complexation information in a wide range of concentration ratios. Copyright 1995, American Chemical Society.