It has long been recognized that islet cell function is rapidly altered in vitro, but can be maintained, at least in part, when cells are layered on defined extracellular matrices. The present work addresses the influence of short-term cell-matrix interactions on islet beta-cell function and provides first insight into the molecular basis of these interactions. When primary rat beta-cells were allowed to attach to a matrix produced by a rat carcinoma cell line (804G), there was an increased insulin secretory response to secretagogues. This change was the result of an increase in the proportion of actively secreting beta-cells and in the amount of insulin secreted per active cell, as shown using the reverse hemolytic plaque assay. In turn, the spreading or flattening of beta-cells on this matrix was enhanced by secretagogues, and flattened cells secreted more insulin than rounded cells. Using indirect immunofluorescence, it was found that 1)alpha6beta1 integrins are present at the surface of islet cells in situ, 2) alpha6beta1 expression is heterogeneous among purified beta-cells and is upregulated by insulin secretagogues, 3) alpha6beta1 expression is higher in spreading cells, and 4) anti-alpha6beta1-specific antibodies decrease spreading. These observations demonstrate that islet cell-matrix interactions can improve the sensitivity of insulin cells to glucose and are mediated, at least in part, by alpha6beta1 integrins, suggesting that outside-in signaling through alpha6beta1 integrin plays a major role in the regulation of beta-cell function.