Phylogeny is often used to compare entire families of genes/proteins. We previously showed that classification of Caenorhabditis elegans Rho GTPases on the basis of their enzymatic properties was significantly different from sequence alignments. To further develop this concept, we have developed an integrated approach to classify C. elegans small GTPases based on functional data comprising affinity for GTP, sub-cellular localization, tissue distribution and silencing impact. This analysis led to establish a novel functional classification for small GTPases. To test the relevance of this classification in mammals, we focused our attention on the human orthologs of small GTPases from a specific group comprising arf- 1.2, evl-20, arl-1, Y54E10BR. 2, unc-108 and rab-7. We then tested their involvement in protein secretion and membrane traffic in mammalian systems. Using this approach we identify a novel network containing 18 GTPases, and 23 functionally interacting proteins, conserved between C. elegans and mammals, which is involved in membrane traffic and protein secretion.