Overexpression and altered trafficking of cathepsins have been associated with the malignant properties of tumors and transformed cells. A characteristic phenotype of transformed cells is also a profound deviation in their metabolism (aerobic glycolysis, glutaminolysis) which enables them to adapt to extreme nutritional conditions. However, whether the altered metabolism may change the expression of proteinases involved in malignancy has not been determined. Herein we present evidences in Kirsten-virus-transformed 3T3 fibroblasts (KBALB) that D-glucose selectively increases active forms of cathepsins L, B, and D, without altering other lysosomal nonproteolytic hydrolases (beta-D-glucosaminidase, acid phosphatase, beta-D-glucuronidase, and beta-D-galactosidase). D-Glucose did not modify mRNA levels for cathepsin B or L and did not affect secretion of pro-cathepsin L. However, D-glucose enhanced strongly the amount of the mature forms of cathepsins B and L, without altering their preferential distribution to light endosomal fractions. Induction by d-glucose of intracellular mature cathepsins B and L required a high growth density of KBALB cells and was reproduced in BALB/3T3 fibroblasts stably transfected with a constitutively activated form of Ras. d-Glucose induction of active cathepsins however was not observed in nontransformed BALB/3T3. D-Mannose, in contrast to nonmetabolized sugars (D-galactose, or L-glucose), caused a similar increase in lysosomal cathepsin activities in dense KBALB cells. The D-glucose analogue, 3-O-methyl-D-glucose, which is transported but not further metabolized, did not reproduce the d-glucose effects. Our findings indicate that, dependent on the nutrient supply and as a consequence of their altered metabolism, transformed cells may modulate the production of active proteinases implicated in malignant progression.