When elicited murine peritoneal exudate cells were subjected to Percoll density gradient centrifugation, polymorphonuclear neutrophils (PMN) were found to distribute over a broad spectrum of buoyant densities (1.10-1.06 g/ml). PMN isolated between approximately 1.10 and 1.085 g/ml were referred to as high density PMN (HD-PMN), and those isolated at approximately 1.085-1.06 g/ml were designated intermediate density PMN (ID-PMN). Cells were characterized on the basis of morphology and specific markers: PMN by lactoferrin immunocytofluorescence and macrophages by nicotinamide adenine dinucleotide glycohydrase activity. Macrophages banded near the top of the gradient with a peak at 1.04 g/ml. At increasing times following elicitation, the ratio of HD to ID-PMN decreased. Decreased density of either murine HD-PMN or human peripheral blood PMN could be induced in vitro by exposure of the cells to endotoxin-activated serum. A decrease in buoyant density of human PMN was also demonstrated in vitro using the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). The response was time dependent, related to dose, and appeared to be mediated by the cell membrane receptor for FMLP. A competitive antagonist of FMLP binding, carbobenzoxy-phenylalanyl-methionine, inhibited the density change with a calculated Kd similar to that reported for inhibition of FMLP-induced aggregation, degranulation, locomotion, and superoxide production. The FMLP-induced decrease in PMN density was shown to be directly correlated with increases in relative mean cell volume. The density response is a new measurement of PMN interaction with specific chemotactic factors, which may be important in the generation of PMN heterogeneity observed in elicited peritoneal exudate cells. In addition, this approach offers a means of physically separating "activated" from "resting" PMN and of studying resultant biochemical differences between these cell populations using both in vivo and in vitro systems.