Class III Semaphorins (SEMA3) comprise a family of chemokines that have been implicated as negative regulators of axonal guidance, angiogenesis and tumor progression. It has been demonstrated previously that one SEMA3, SEMA3F, may have therapeutic potential in the treatment of cancer. When transfected with SEMA3F, the highly metastatic human melanoma cell line A375SM was found to exhibit a highly-encapsulated, avascular phenotype with limited metastasis. Members of SEMA3 are regulated on many levels, including proteolytic processing. SEMA3F, like other SEMA3, is expressed as a 100 kD proprotein that is seen to be processed in vitro and in vivo to 95 and 65 kD isoforms. This has been largely attributed to furin-like endoproteases on the basis of furin inhibition studies. However, currently available small chemical or peptide inhibitors against the family of subtilisin/kexin-type proprotein convertases (PCSK), to which furin belongs, do not have good selectivity between PCSKs. Cleavage of SEMA3 to 65 kD have been shown to have differing effects. SEMA3A loses its ability to repel sympathetic ganglia and SEMA3E reverses its phenotype from chemorepulsant to chemoattractant for developing vasculature following cleavage. In order to further develop therapeutic strategies based on SEMA3F, it is therefore critical to better understand the proteolytic regulation of this molecule. In this study, it is shown that digest of purified SEMA3F with purified recombinant human furin does not result in proteolytic cleavage and suggested that the cleavage of SEMA3F to a 65 kD isoform may be mediated by other members of the PCSK family.