The role of glycosylation in the synthesis, transport, and localization of procathepsin L has been analyzed. Procathepsin L is the precursor form of a lysosomal cysteine protease which is synthesized in large amounts by malignantly transformed cells and cells treated with growth factors or tumor promoters. As for other lysosomal hydrolases, procathepsin L is predominantly secreted when it is overexpressed, despite the presence of the lysosomal sorting marker mannose 6-phosphate. To study the role of glycosylation in procathepsin L trafficking, we constructed a cDNA in which the codon for Asn-204 was mutated to encode Gln. When this mutated cDNA was transfected into NIH 3T3 cells, a completely nonglycosylated form of procathepsin L was expressed. The protein appeared to be folded properly, as judged by its ability to be proteolytically processed to a lower molecular weight form during incubation at pH 4. Nonglycosylated protein was stable, both as unprocessed proenzyme and after processing at pH 4, and it was predominantly secreted instead of being delivered to lysosomes. Nonglycosylated and endogenous glycosylated procathepsin L were secreted by NIH 3T3 cells with identical kinetics. These studies therefore confirm that Asn-204 is the normal functional carbohydrate acceptor in procathepsin L and that carbohydrate in wild-type procathepsin L serves predominantly as a lysosomal targeting signal, with little or no role in protein folding or stability. Furthermore, there does not appear to be a major mannose 6-phosphate-independent route of lysosomal localization in NIH 3T3 cells. In experiments with a second mutant, the glycosylation signal at Asn-251, which normally is not utilized, was capable of serving as a carbohydrate acceptor, suggesting that there is normally a structural impediment to glycosylation at Asn-251 in procathepsin L.