The number of neutrophils, eosinophils, and mononuclear cells that had migrated into pouch fluid were measured after injecting an antigen (azobenzenearsonate-conjugated acetyl bovine serum albumin) solution into air pouches made on the dorsum of immunized rats. The number of neutrophils began to increase 4 hr after the antigen challenge, reaching a maximum at 16-24 hr. Increases in the number of eosinophils and mononuclear cells were quite poor. The pouch fluid supernatant fraction was fractionated to hydrophilic and lipophilic fractions with the aid of an octadecylsilyl silica cartridge, and the chemotactic activity in each fraction was measured by the modified Boyden chamber method. The chemotactic activity in the pouch fluid supernatant fraction was the highest at 2-4 hr and decreased with time after the antigen challenge. At 16 and 24 hr, chemotactic activity was decreased to a very low level although there was a large number of neutrophils in the pouch fluid. At 4 hr, the chemotactic activity in the lipophilic fraction was significantly higher than that in the hydrophilic fraction. However, at 8 hr, the chemotactic activity in the lipophilic fraction was changed to a significantly lower level than that in the hydrophilic fraction. The chemotactic activity in the lipophilic fraction had decreased to a very low level at 8 hr. Heat treatment of the pouch fluid supernatant fraction at 92 degrees C for 5 min both at 4 and 8 hr significantly increased the chemotactic activity.(ABSTRACT TRUNCATED AT 250 WORDS)