Since the number of polymorphonuclear leukocytes (PMN) responding to a chemotactic stimulus in the Boyden chamber is influenced by the cell density, we studied whether this variable was important in determining chemotaxis under agarose. The chemotactic index was determined by summing the product of each cell that had migrated from the chemotactic well by its distance and correcting this sum for random migration. Cell density (number of PMN per mm2 surface area of the agarose plate well) influenced the number of cells responding to the chemotactic stimulus. Only when more than 1.8 x 10(2) PMN/mm2 were used was chemotaxis then detected. For cell densities greater than this number, there was a highly positive correlation between cell density and chemotactic index (P less than 0.001). These findings are consistent with previous reports and indicate that PMN density is a critical variable when using the agarose method. In addition, these studies provide further evidence for cellular cooperation in the initial phases of the chemotactic response.