To investigate eosinophil stimulation by chemokines we developed a sensitive assay of leukocyte shape change, the gated autofluorescence/forward scatter assay. Leukocyte shape change responses are mediated through rearrangements of the cellular cytoskeleton in a dynamic process typically resulting in a polarized cell and are essential to the processes of leukocyte migration from the microcirculation into sites of inflammation. We examined the actions of the chemokines eotaxin, eotaxin-2, monocyte chemoattractant protein-1 (MCP-1), MCP-3, MCP-4, RANTES, macrophage inflammatory protein-1α (MIP-1α), and IL-8 on leukocytes in mixed cell suspensions and focused on the responses of eosinophils to CC chemokines. Those chemokines acting on CCR3 induced a rapid shape change in eosinophils from all donors; of these, eotaxin and eotaxin-2 were the most potent. Responses to MCP-4 were qualitatively different, showing marked reversal of shape change responses with agonist concentration and duration of treatment. In contrast, MIP-1α induced a potent response in eosinophils from a small and previously undescribed subgroup of donors via a non-CCR3 pathway likely to be CCR1 mediated. Incubation of leukocytes at 37 C for 90 min in the absence of extracellular calcium up-regulated responses to MCP-4 and MIP-1α in the majority of donors, and there was a small increase in responses to eotaxin. MIP-1α responsiveness in vivo may therefore be a function of both CCR1 expression levels and the regulated efficiency of coupling to intracellular signaling pathways. The observed up-regulation of MIP-1α signaling via non-CCR3 pathways may play a role in eosinophil recruitment in inflammatory states such as occurs in the asthmatic lung.