Collagen gel contraction assay is a method for evaluating contraction of cells embedded in collagen gel matrices through measuring the gel size. In the present study, we established a protocol for collagen gel contraction assay using human bronchial smooth muscle cells obtained commercially, and applied it for evaluation of inhibitory effect of formoterol on histamine-induced contraction. Human bronchial smooth muscle cells were embedded in collagen gel in wells of 24-well plates, and gel contraction against histamine or acetylcholine was observed. Gel size was measured at an interval of 10 min for 60 min from the addition of a stimulant. Both acetylcholine and histamine caused gel contraction in a concentration-dependent manner and the contraction by histamine was apparently potent than that by acetylcholine. Formoterol at concentrations of 10⁻¹⁰–10⁻⁷ M inhibited collagen gel contraction caused by histamine concentration-dependently. Pre-treatment with fluticasone at a concentration of 10⁻⁸ M apparently potentiated the inhibitory effect of formoterol at 10⁻¹⁰ and 10⁻⁸ M on collagen gel contraction by histamine. Prolonged pre-treatment with 10⁻⁸ M formoterol abolished the inhibitory effect of 10⁻⁸ M formoterol. Furthermore, 4 h simultaneous pre-treatment with 10⁻⁸ M formoterol and fluticasone partially but significantly recovered the inhibitory effect of 10⁻⁸ M formoterol. Present results indicate that the collagen gel contraction assay using human bronchial smooth muscle cells is useful for evaluating the effects of bronchodilating drugs, and that fluticasone potentiates the inhibitory effect of formoterol on histamine-induced collagen gel contraction.