It is commonly assumed that the sum toto of chemotaxis is responsiveness to spatial gradients of attractants. However, in the model system Dictyostelium, in which the signal was elegantly visualized over 20 years ago by Peter Devreotes and colleagues, the signal is in the form of a symmetric, nondissipating wave of cAMP relayed outwardly through a natural population of aggregating cells. This wave has temporal and concentration components, as well as spatial components, that all cue cell behavior in the chemotactic process. In the front of each wave, each cell experiences an increasing temporal gradient and positive spatial gradient of cAMP; at the peak of each wave, each cell experiences a cAMP concentration inhibitory to locomotion; and in the back of each wave, each cell experiences a decreasing temporal and negative spatial gradient of cAMP. Cells respond in a unique and specific fashion to each of the four phases of the wave, and this sequence of behaviors together represents the chemotactic response. A defect in response to any one of the components of the wave compromises natural chemotaxis. Hence, mutants that can efficiently respond to a spatial gradient, but not a temporal gradient cannot chemotax in nature (i.e., in the natural wave). Protocols are described to analyze the basic motile behavior of cells in the absence of a chemotactic signal, and to test the responsiveness of cells to the individual temporal, spatial and concentration components of a natural wave. These protocols include computer-assisted methods for analyzing cell behavior developed in the Keck Dynamic Image Analysis Facility at Iowa. The results of mutant analyses using these protocols have been used to develop realistic models of cell motility and chemotaxis. These models include independent parallel regulatory pathways emanating from different phases of the wave, each affecting a different phase-specific behavior. Results will be presented demonstrating that human PMNs possess all of the machinery necessary to read every aspect of a wave, like Dictyostelium does, suggesting that signals in the human body may be in the form of relayed waves. Finally, an example will be presented of the successful application of this approach to a human disease, the Shwachman-Diamond Syndrome.