Desensitization of the neutrophil inflammatory response to intracutaneous injection of chemotaxins and endotoxin was studied in rabbits. When restimulated 6 hr later with the same agent, inflammatory lesions initiated with platelet-activating factor (PAF), leukotriene B4 (LTB4), or endotoxin supported a diminished influx of neutrophils compared with responses in normal skin. In contrast, repeated stimulation of lesions with alpha-casein failed to lead to desensitization. The specificity of desensitization was investigated, and it was found that lesions initiated with formylmethionyl-leucyl-phenylalanine (FMLP) supported a normal response when restimulated with PAF, alpha-casein, or endotoxin. Initiation of lesions with LTB4, however, diminished the subsequent response to zymosan-activated plasma and PAF, but not endotoxin, alpha-casein, or FMLP. This result indicates that LTB4 is not a final common mediator of neutrophil infiltration of acute inflammatory lesions. Desensitization was detected irrespective of the concentration of chemotaxin used to investigate the response. Repeated stimulation of lesions with FMLP abolished the accumulation of neutrophils after the final stimulus, indicating that complete desensitization can occur and the presence of a chemotaxin within an inflammatory lesion is not sufficient stimulus for neutrophil infiltration of the site to proceed. Lesions initiated with endotoxin supported comparable responses when restimulated with a mixture of FMLP and endotoxin or FMLP alone, despite 93% inhibition of the response to restimulation with endotoxin alone. This indicates that a cell-directed inhibitor of neutrophil migration, such as lipomodulin or neutrophil-immobilizing factor, does not produce the diminished responses in desensitized lesions. It is proposed that desensitization occurs due to down-regulation of a receptor-coupled pathway that permits or facilitates neutrophil extravasation in acute inflammatory lesions. The chemotaxin receptors regulating neutrophil extravasation are probably located on endothelial cells of post-capillary venules.