The accumulation of polymorphonuclear leukocytes (PMNs) was measured in rabbits following single or repeated injections into skin sites of the inflammatory agents, zymosan-activated plasma and formylmethionyl-leucyl-phenylalanine. Fewer cells entered lesions restimulated with the same agent during the subsequent inflammatory response than simultaneously entered skin sites stimulated for the first time. Decreased reactivity of a restimulated site was specific to the initiating stimulus, developed within 2 to 4 h of initial stimulation and persisted for at least 8 h. The results indicate that PMN accumulation in acute inflammatory lesions is regulated locally by a chemotactic factor-specific mechanism which differs from PMN deactivation and concentration gradient mechanisms currently thought to regulate PMN migration in vivo.