Abstract The chemotactic properties of homogeneus human C5a and C5a des Arg, as well as other cytotaxins, can be evaluated accurately by using a modified ‘chemotaxis under agarose’ method that requires neither serum nor albumin. Gelatin, at a final concentration of 0.25%, substitutes adequately for the serum or albumin that has been used previously. By assessing both the directed and random migration of human neutrophils under a variety of conditions, we delineated characteristic features of the neutrophils' chemotactic response to 3 cytotaxins: C5a, C5a des Arg and N-f-Met-Phe. Neutrophils responding to these stimuli move across the glass microscope slide that serves as a supporting surface in 3 distinctly different migratory patterns designated here as ‘rocket-shaped, blunt and desensitized’. These readily identifiable patterns each correlate with a unique neutrophil morphology and depend both on the nature and the concentration of the chemotactic factor present. From the distance the neutrophils migrate after various time intervals, we can calculate the mean initial rate of migration for the most rapidly moving cells. These studies show that human neutrophils migrate at an average initial rate of 5.0 ± 0.2 μ/min in response to each of the factors tested and at an average random rate of 1.7 ± 0.1 μ/min in the absence of any chemotactic stimulus. Optimum chemotaxis, as judged by ‘chemotactic index’ values, occurs when a total of 2.5 × 10 5 neutrophils/10 μl is incubated for 2–2.5 h at 37°C. This modified assay is sufficiently sensitive for use in assessing the chemotactic properties of a wide variety of factors. In particular, it may become the preferred method for studying purified human C5a and C5a des Arg because the latter factor fails to stimulate neutrophil migration in the Boyden chamber assay system in the absence of serum or serum proteins.