The switch of human melanoma cell phenotype from non to highly tumorigenic and metastatic is triggered by the increase of procathepsin L secretion, which modifies the tumour microenvironment. The aim of the present study was to identify components involved in the regulation of procathepsin L secretion in melanoma cells. We focused on Rab family members, i.e. Rab3A, Rab4A, Rab4B, Rab5A, Rab8A, Rab11A, Rab27A and Rab33A, which are involved in distinct regulatory pathways. From analysis of mRNA and protein expression of these Rab components and their knockdown by specific siRNAs (small interfering RNAs) it emerged that Rab4A protein is involved in the regulation of procathepsin L secretion. This result was strengthened as procathepsin L secretion was either inhibited by expression of a Rab4A dominant-negative mutant or increased by overexpression of the wild-type Rab4A. Rab4A regulation: (i) discriminates between procathepsin L secretion and expression of intracellular cathepsin L forms; (ii) did not modify other Rab proteins and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression, or IL-8 (interleukin-8) and MMP-2 (matrix metalloproteinase-2) secretion; and (iii) was still efficient during unglycosylated procathepsin L secretion. Thus down- or up-regulation of Rab4A expression or Rab4A function triggered inhibition or increase of procathepsin L secretion respectively. Furthermore, Rab4A regulation, by modifying procathepsin L secretion, switches the tumorigenic phenotype of human melanoma cells in nude mice.