A chlorodiazirine derivative of pentamidine was synthesized and tested for anti-trypanosomal activity using EATRO stock 164 trypanosomes in cell culture. Anti-trypanosomal activity was measured as a decrease in [³H]hypoxanthine incorporation by the organisms. The derivative, 3,3′-[1,5-pentanediylbis(oxy-4,1-phenylene)]bis(3-chloro-3H-diazirine), at a treatment level of 0.1 µM inhibited isotope incorporation by 40–50% compared to nontreated controls. At this concentration, pentamidine inhibited incorporation only 10–15%. The derivative is a nonionic molecule with much different solubility properties than the parent compound and should readily cross the blood–brain barrier.