Sef (similar expression to fkf genes) was identified as an inhibitor of FGF signaling. The regulation of this inhibitory effect was largely unknown. In this report we demonstrated that tyrosine 330 in hSef protein plays a critical role in the control of the protein localization and thereby in the regulation of Ras/MAPK signaling pathway. We found that the tyrosine 330 is in the form of the YXX Phi signal context and mutation of this residue resulted in preferred plasma membrane localization of hSef. We also observed that both Sef and SeFY330F (where tyrosine is substituted by phenylalanine) interacted and co-localized with FGFR in the co-immunoprecipitation assay, and immunostaining assay respectively. We further revealed that the increased amount of Sef localization in the plasma membrane was coupled with the enhanced inhibitory effect on the FGF signaling pathway, indicating that Sef might exert its inhibitory function on the plasma membrane. This paper revealed that tyrosine 330 is critical for the inhibitory function of Sef on FGF signaling. (c) 2007 Elsevier Inc. All rights reserved.