Many bacterial species express tyrosine kinases termed BY-kinases that share no homology with eukaryotic enzymes. We have previously reported that the Staphylococcus aureus BY-kinase CapB2 when fused with the C-terminal activation domain of its modulator CapA1, can translate into an active tyrosine kinase in HEK293T cells. In the present study, full-length CapA1 and CapB2 tagged with different fluorescent proteins were transfected into HEK293T cells. When expressed individually, the modulator CapA1, a membrane protein in bacteria, also appeared to localize to the cell membrane in HEK293T cells. In contrast, the catalytic subunit, CapB2, was found to be cytosolic. Coexpression of the two proteins resulted in apparent translocation of CapB2 to the membrane with concomitant activation of tyrosine kinase activity. This translocation and activation of CapB2 did not occur when the cytoplasmic C-terminal tail of CapA1 was deleted. Conversely, the CapA1 cytoplasmic C-terminal tail alone, when attached to a membrane localization sequence, was sufficient for CapB2 translocation and kinase activation. Our results indicate that the kinase activity of CapB2 is stimulated by direct interaction with the C-terminal cytoplasmic domain of CapA1 and that the process can be reconstituted and visualized in a human cell line. We created various mutants of CapA and CapB, and present data that demonstrate the correlation between CapA-CapB interaction and kinase activation.