Fatty acids induce cholecystokinin (CCK) secretion both in humans and from murine enteroendocrine cell lines. In both cases, only fatty acids above a critical acyl chain length (C₁₀) are capable of inducing a response. Using the enteroendocrine cell line STC‐1, the aim of this study was to determine whether this acyl chain length dependency is related to the fact that longer chain fatty acids are relatively insoluble in aqueous solutions and, if so, whether it is insoluble aggregates of fatty acids rather than free fatty acids which evoke CCK secretion. Solutions of fatty acids (chain length C₈–C₁₄), which were judged by filtration and Zeta sizer measurement to contain no fatty acid aggregates, never evoked CCK secretion from STC‐1 cells. Filtering fatty acid solutions (of chain length C₁₀, C₁₂ and C₁₄) through polytetrafluoroethylene (PTFE) filters (0.45 μm pore size) revealed a narrow concentration range for each acid over which the amount of fatty acid removed from the solution increased sharply due to the formation of fatty acid aggregates. Filtration experiments, in which suspensions of C₁₀, C₁₂ and C₁₄ fatty acids were passed through pore sizes of 0.2, 0.45 or 1.2 μm, suggested that STC‐1 cells did not respond to fatty acid aggregates of greater than 1.2 μm, while at least 50 % of the CCK response was mediated by aggregates which were smaller than 0.45 μm. Fatty acids induce CCK secretion from STC‐1 cells by elevating intracellular Ca²⁺ concentration ([Ca²⁺]_i). We therefore measured the effects on [Ca²⁺]_i of filtered C₁₀, C₁₂ and C₁₄ fatty acids. In all cases, [Ca²⁺]_i responses were closely correlated with CCK secretion. Interestingly, while filtrates of fatty acid solutions evoked CCK secretion and elevated [Ca²⁺]_i, freshly prepared solutions of fatty acids at the same concentration as the filtrates did not. This suggested that fatty acid aggregates were not in equilibrium with the solvent after filtration. The observation that the ability of C₁₀, C₁₂ and C₁₄ filtrates to elevate [Ca²⁺]_i decayed with time was consistent with this hypothesis. Furthermore, sonication of the filtrates abolished their ability to elevate [Ca²⁺]_i. These data further suggest that it is a physical property of the fatty acid solution (the presence of insoluble fatty aggregates) which is responsible for the observed cellular responses. We conclude that Ca²⁺ mobilisation and CCK secretion in STC‐1 cells is driven by a signal transduction mechanism that senses insoluble fatty acid aggregates, rather than free fatty acids in solution.