Classical late infantile neuronal ceroid lipofuscinosis is an autosomal recessive disease caused by mutations in the CLN2 gene resulting in functional defects of the gene product tripeptidyl-peptidase I. This disease is associated with a progressive neurodegenerative course beginning at the age of two years with developmental stagnation, finally leading to a complete loss of motor function, vision and speech by the age of 10 years. We analyzed the functional consequences of the mutations R127Q, R208X, N286S, I287N, T353P and Q422H, which were previously identified in patients with late infantile ceroid lipofuscinosis, with regard to enzymatic activity, stability, post-translational processing and intracellular localization of tripeptidyl-peptidase I. We could not detect any translational product for the mutant R208X. We found that four missense mutations, N286S, I287N, T353P and Q422H, which are located in conserved protein regions of tripeptidyl-peptidase I, decreased the enzymatic activity dramatically, blocked processing to mature size peptidase and led to protein retention in the endoplasmatic reticulum and rapid degradation in non-lysosomal compartments. We conclude that these amino-acid substitutions induce major misfolding of the precursor peptidase and hence prevent post-translational processing and lysosomal targeting of tripeptidyl-peptidase I. In contrast, the amino-acid substitution R127Q within a non-conserved protein region did not significantly affect enzymatic activity, stability, processing and lysosomal targeting of tripetidyl-peptidase I. Thus, our functional analyses of CLN2 mutations reveal novel insight into the molecular defect underlying dysfunction of tripeptidyl-peptidase I.