Chemotaxis of human leukocytes was studied in vitro with a microfilter having pores of 3 micrometer used as a substrate for the gradient. Under these conditions, nonsegmented neutrophils did not reach the compartment filled with the attractant, but a significant proportion of them did so when filters with larger pores were substituted. When leukocytes from infected patients were tested with the usual 3 micrometer pore filters, chemotaxis was reduced (as previously shown), but less markedly and less frequently in simultaneous experiments with larger pores. In experiments performed under agarose layers instead of filters, nonsegmented neutrophils responded normally to chemoattraction, again suggesting that their impaired migration in filter experiments was a matter of pore size. When leukocytes from infected patients were assayed under agarose, no impairment occurred; on the contrary, a slight increase in both chemotaxis and random motility was observed. It was therefore concluded that some published cases of impaired neutrophil chemotaxis in infection might be due to technical bias related to pore size.