The large subfamily of receptor tyrosine kinases (RTKs) for which EPH is the prototype have likely roles in intercellular communication during normal mammalian development, but the biochemical signalling pathways utilised by this family are poorly characterised. We have now identified two in vitro autophosphorylation sites within the juxtamembrane domain of the Eph family member Sek, and a candidate binding protein for the activated Sek kinase. Specific antibodies defined Sek as a 130 kDa glycoprotein with protein kinase activity expressed in keratinocytes, whilst a bacterially expressed gst-Sek kinase domain fusion protein autophosphorylated exclusively on tyrosine residues, confirming that Sek encodes an authentic protein tyrosine kinase. Two dimensional phosphopeptide mapping and site-directed mutagenesis defined juxtamembrane residue Y602 as a major site of in vitro autophosphorylation in Sek, whilst Y596 was phosphorylated to a lower stoichiometry. Complimentary approaches of in vitro binding assays and BIAcore analysis revealed a high affinity association between the Y602 Sek autophosphorylation site and the cytoplasmic tyrosine kinase p59fyn, an interaction mediated through the SH2 domain of this intracellular signalling molecule. Moreover, these data identify the novel phosphotyrosyl motif pYEDP as mediating high affinity association with fyn-SH2, extending the previously defined consensus motif for this interaction. The extensive conservation of this fyn-binding motif within the juxtamembrane domain of Eph family RTKs suggests that signalling through fyn, or fyn-related, tyrosine kinases may be utilised by many members of this large subclass of transmembrane receptors.