The aim of this study was to establish the cause of insensitivity of T(84) human colonic epithelial cells to 5-hydroxytryptamine (5-HT). Monolayers of T(84) cells were placed in modified Ussing chambers for measurement of short-circuit current, an index of secretion. When grown in serum-supplemented media, T(84) cells gave secretory responses to acetylcholine and forskolin but not to 5-HT. When grown in AIM V serum-free media, T(84) cells responded to 5-HT. Chromatographic analysis with fluorimetric detection showed high levels of 5-HT (1.8 microM) in the serum. This contamination is probably responsible for subsequent desensitization of T(84) cells to 5-HT.