To investigate the role of neutrophils in the efficacy of photodynamic therapy (PDT) in rhabdomyosarcoma-bearing rats, the number of these circulating phagocytes was decreased or increased before interstitial PDT by use of rabbit anti-rat neutrophil serum or granulocyte colony-stimulating factor, respectively. After administration of the antiserum, the number of circulating neutrophils decreased by 99.9%. However, the number of monocytes, lymphocytes, and platelets decreased as well (by 100%, 80%, and 25%, respectively). Under these conditions, PDT did not retard tumor growth at all. However, after cessation of the antiserum treatment 5 days after PDT, a striking decrease in the growth rate occurred subsequent to an increase above the normal range of the number of circulating neutrophils. Administration of the granulocyte colony-stimulating factor led to a specific 4-fold increase in the number of circulating neutrophils. In these rats, the tumor growth at day 2 after PDT was retarded as compared with PDT-treated rats that received saline only. Statistical evaluation of both experimental conditions showed that the efficacy of PDT, expressed as the percentage of change in tumor volume at day 2 after treatment, was dependent on the number of circulating neutrophils present at the day of PDT (P = 0.001; r2 = 0.482). Apparently, neutrophils are indispensable for successful PDT in vivo.