The chemical structure of an unknown metabolite of ciprofloxacin (CAS 85721-33-1) is characterized by means of reversed phase ion pair liquid chromatography, absorption and fluorescence spectroscopy, partition coefficients as well as chemical and enzymatic hydrolytic degradation. A chemical structure of the unknown metabolite is proposed: N-formyl-desethylen-ciprofloxacin. It can be formed as an intermediate in the oxidative degradation of ciprofloxacin via oxociprofloxacin to desethylen-ciprofloxacin, or it may be formed by conjugation of desethylen-ciprofloxacin with formic acid. The amounts found in plasma and urine of patients were in the range of desethylen-ciprofloxacin, i.e. about 1% of the parent compound.