A cDNA clone for a physiologically regulated Tetrahymena cysteine protease gene was sequenced. The nucleotide sequence predicts that the clone encodes a 336-amino acid protein composed of a 19-residue N-terminal signal sequence followed by a 107-residue propeptide and a 210-residue mature protein. Comparison of the deduced amino acid sequence of the protein with those of other cysteine proteases revealed a highly conserved interspersed amino acid motif in the propeptide region of the protein, the ERFNIN motif. The motif was present in all of the cysteine proteases in the data base with the exception of the cathepsin B-like proteins, which have shorter propeptides. Differences in the propeptides and in conserved amino acids of the mature proteins suggest that the ERFNIN proteases and the cathepsin B-like proteases constitute two distinct subfamilies within the cysteine proteases.