The involvement of Ras and three major Ras effectors, Raf, phosphatidylinositol 3-kinase (PI3K) and Ral guanine nucleotide exchange factor in the regulation of lysosomal proteases cathepsin L and B in human fibroblasts was compared. We found that cathepsin L cell content was increased by active Ras overexpression through Raf- and PI3K-mediated signaling pathways, while cathepsin B processing was altered by active Ras overexpression. Cathepsin L increased level following active Ras overexpression correlates with an increase of p38 MAPK activation and content and with an increase of p44/42 MAPK activation, so we investigated the role of these signaling pathways using pharmacological inhibitors. Unexpectedly, the p38 MAPK inhibitor SB203580 produced an increase of cathepsin L content, while the p44/42 MAPK signaling cascade inhibitor U0126 produced a remarkable shift of cathepsin L processing in favor of procathepsin L. In both cases, cathepsin B level and processing were not affected. The analysis of CTSL1 gene transcript demonstrated that cathepsin L protein and transcript correlate both in fibroblasts expressing Ras mutants and in pharmacologically treated cells, thus indicating a transcriptional up-regulation.