The yeast aspartic protease, proteinase A, has a 54 amino-acid propeptide, which is removed during activation of the zymogen in the vacuole. Apart from being involved inhibition/activation, the propeptide has been shown to be essential for formation of a stable active enzyme (van den Hazel, H. B., Kielland-Brandt, M. C., and Winther, J. R. (1993) J. Biol. Chem. 268, 18002-18007). We have investigated the sequence requirements for function of the propeptide. The N-terminal half and the C-terminal half of the propeptide were replaced by random sequences at the genetic level, and collections of the mutants were subjected to a colony screen for ones exhibiting activity. A high frequency (around 1%) of active constructs was found, which indicates a very high tolerance for mutations in the propeptide. Thirty-nine functional mutant forms containing random sequence at either the N- or C-terminal half of the propeptide were characterized. Comparison of the propeptides of the active constructs suggests that a particular lysine residue is important for efficient biosynthesis of proteinase A.