In this study, the effects of chronic wound fluid on the structure and biological activity of becaplermin (recombinant human platelet-derived growth factor–BB [rhPDGF-BB]) were evaluated. Wound fluid was collected from 12 subjects with diabetic ulcers or pressure ulcers. Wound fluid ± becaplermin was added to cell cultures before- and after incubation for 12 hours at 37°C or after 12 hours’ topical treatment. Biological activity, concentration, and immunogenicity were determined by [³H]thymidine incorporation into quiescent human foreskin fibroblasts, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis, respectively. No PDGF-BB or mitogenic activity was detected in chronic wound fluid alone. Mitogenic activity was present in post-treatment samples from becaplermin-treated subjects but not placebo-treated subjects. Exposure to chronic wound fluid for 12 hours did not alter the amount, banding pattern, or mitogenic activity of becaplermin. Biologically active becaplermin remains in wound fluid 12 hours after topical application of becaplermin gel.