T he chemotactic migration of leukocytes is preceded by an alteration in the cells' shape from round to a characteristic polar configuration. We have developed an assay that shows that human monocytes, when exposed to chemoattractant in suspension, assume this polarized shape. The three types of chemo-attractants studied, a chemotactic lymphokine, complement-activated serum, and the N-formylated oligopeptides, all induced polarization in a time, temperature, and dose-dependent fashion. Nonchemotactic agents such as mitogens or phorbol myristate acetate did not induce polarization. At 37°C, polarization was rapid (<1 min) and was inhibitable by cytochalasin B, sodium azide, or low temperature. A series of N-formylated oligopeptides were studied and their activity in inducing polarization correlated closely (r > 0.99) with their chemotactic activity. Of the entire population of circulating monocytes there is a subpopulation of cells that is capable of polarizing in response to chemotactic stimuli. The maximum percentage of monocytes which polarized to any chemotactic factor was ∼60%. Furthermore, the combination of several chemotactic factors could not increase the percentage of polarized monocytes above the maximum obtained with an optimal dose of any single chemoattractant. The data also demonstrate that high doses of a chemoattractant can induce a state of cross-desensitization in monocytes that blocks the response of the cells to other types of chemotactic factors. These results support the concept that the monocytes that do respond to chemotactic stimuli are capable of responding to any of several attractants.