An inactive cathepsin B-like enzyme with a molecular mass of 40 kD was found in a human melanoma culture medium. The inactive form of this enzyme was converted into an active form with a molecular mass of 28 kD by pepsin treatment. This activated cathepsin B-like enzyme had almost the same characteristics regarding molecular size, substrate specificity, dependence on chemical reagents, and Km values as intracellular cathepsin B. Sodium dodecylsulphate polyacrylamide gel electrophoresis followed by electroblotting with an antiserum against cathepsin B yielded inactive cathepsin B-like enzyme fractions which showed two immunoreactive bands with molecular masses of 40 and 28 kD, respectively. On the other hand, alkali treatment of the inactive cathepsin B-like enzyme fractions released a cysteine proteinase inhibitor with a molecular mass of 12 kD. These data suggest that these inactive cathepsin B-like enzymes in melanoma culture medium are present not only in the precursor form, but that they are also present as enzyme-inhibitor complexes, both of which can be activated enzymatically in vitro.