The effects of chemotactic factors on rabbit neutrophils were evaluated measuring cell migration in modified Boyden chambers and under agarose, in lysosomal enzyme release, leukocyte aggregation, and in vivo neutropenia. Chemotactins employed included the complement-derived C3 and C5 fragments, the bacterial chemotactic factor from culture supernatant fluids of Escherichia coli, and the synthetic chemotactic factors Met-Leu-Phe and formyl-Met-Leu-Phe. A consistent parallelism was found in all the leukocyte responses to a given chemotactic factor. In no instance, with any of the five chemotactic factor preparations, did cells responding in one assay system fail to respond in the four other assay systems, suggesting a common event in all of the cell responses. Boyden chamber chemotaxis was consistently the most sensitive assay; the agarose assay was, in general, less sensitive by a factor of 100 fold. Enzyme release approached, in cell sensitivity to chemotactic factors, that of the Boyden chamber assay. In general, in vitro leukocyte aggregation and in vivo neutropenia were considerably less sensitive assays. Chemotactic factor inactivator (CFI) purified from human serum destroyed in parallel all biological activities of C3 and C5 chemotactic factors but had no effect on the bacterial chemotactic factor and the activities of synthetic chemotactic peptides.