We have previously reported on the release of neutrophil chemotactic factors (NCF) from injured conjunctival tissue. The present study was designed to biochemically characterize these conjunctiva-derived chemotactic factors and determine their biological activities. Bulbar conjunctiva was surgically removed from a rabbit eye and incubated with 250 microL of minimal essential medium (MEM) for 6 hours at 37 degrees C in a 5% CO2 atmosphere. Chemotactic activity was assayed using modified Boyden chambers with rabbit peritoneal neutrophils as indicator cells. Following treatment with subtilisin protease for 90 minutes, chemotactic activity of the conjunctival factors was reduced by 74%. Similarly, activity was lost after heating at 56 degrees C for 60 minutes (41% inhibition). Using ultrafiltration techniques, we showed that the majority of the chemotactic activity remained above a 100 kilodalton filter, suggesting the existence of high molecular weight factors. We also showed that the conjunctival factors are not glycoproteins and bind to both anion and cation exchange resins. When 100 microL of conjunctival supernatant was injected in the superior tarsal conjunctiva of rabbits, significant recruitment of neutrophils was evident by 4 hours. Control rabbits injected with MEM did not show neutrophil recruitment. Results of these studies indicate that NCF from traumatized conjunctival tissue are proteins (and not glycoproteins) of high molecular weight, heat labile, exhibit anionic and cationic charges, and are active in vivo.