We found that when first encountering with an ECM-patterned substrate, fish keratocytes can selectively adhere and migrate along the FBN paths only for a limited period of time, then the cell��s response to substrate guidance becomes adaptive or desensitized. Thirty min after plating, most fish keratocytes have moved out of the FBN path confinement and are undergoing undirected random migration. Interestingly, a calcium transient created by the calcium uncaging technique, or indeed from the native intracellular calcium Etodolac oscillations, can re-sensitize an adaptive cell and render it responsive, for another short period of time, to substrate ECM guidance. Mechanisms underlying the adaptation and the calcium-induced CASIN resensitization-desensitization process are explored. Many experimental approaches were tried with the aim of converting fish keratocyte��s adaptive random migration into ECM path-guided movement. We found that a burst increase in intracellular calcium created by calcium uncaging, when applied to a single fish keratocyte using a focused laser beam or to a selected population of cells using wide-field illumination, could successfully change the undirected motion into ECM-guided movement. As shown in Fig. 1A, uncaging done at 0:00 to the keratocytes that were already adaptive to the patterned ECM substrate enabled many of them to resensitize and response to substrate guidance again by preferentially spreading and migrating along the FBN path. Such calcium-induced resensitization to substrate guiding cues was transient; the resensitized cells later became desensitized and moved out of the FBN path and underwent undirected migration. During such a calcium-induced resensitization-desensitization process, the trend of a transient increase followed by a decrease in the number of FBN path-associated cells was obvious for the sequence of Movie S1, and for averaged values obtained from three independent experiments. Under high magnification and calcium imaging microscopy, we noticed an immediate halt of undirected cell movement right after the single controlled release of intracellular “calcium burst”.