The influence of hemoglobin on the chemotactic response of human polymorphonuclear neutrophils (PMNs) and monocytes under agarose was evaluated. Hemoglobin present at the site of the chemotactic factor, E. coli culture filtrate (BFE) or zymosan-activated serum (ZAS), in a concentration of 4% causes depression of the chemotactic response of cells of both types. The degree of depression of the response is directly proportional to the concentration of hemoglobin (2–10%) at the site of chemotactic factor. If hemoglobin is added to the cells, much lower concentrations (0.001–0.1%) are sufficient to elicit the same effect. Hemoglobin did not interfere with the spontaneous migration of the cells in this experiment unless its concentration at the site of the cells was higher than 0.1%. The presented evidence supports the idea that hemoglobin inhibits the chemotactic response of PMNs by interfering with the effective interaction of the cytotaxin to the receptor on the cell wall. These findings are consistent with the findings that the influx of PMNs into the peritoneal cavity in response to intraperitoneal bacterial inoculum is inhibited by hemoglobin and that the mechanism by which PMNs are prevented from responding to bacteria in vivo involves the interference by hemoglobin with the natural chemotactic response of PMNs.