The motor protein kinesin is a heterotetramer composed of two heavy chains of ~120 kDa and two light chains of ~6 kDa protein. Kinesin motor activity is dependent on the presence of ATP and microtubules. The kinesin light chain-binding site in human kinesin heavy chain was determined by reconstituting in vitro a complex of recombinant heavy and light chains. The proteins expressed in bacteria included oligohistidine-tagged fragments of human ubiquitous kinesin heavy chain, spanning most of the stalk and all of the tail domain (amino acids 555-963); and untagged, essentially full-length human kinesin light chain (4-569) along with N-terminal (4-363)and C-terminal (364-569) light chain fragments. Heavy chain fragments were attached to Ni2+-charged beads and incubated with untagged light chain fragments. Analysis of eluted complexes by SDS-PAGE and immunoblotting mapped the light chain-binding site in heavy chain to amino acids 771-813, a region close to the C-terminal end of the heavy chain stalk domain. In addition, only the full-length and N-terminal kinesin light chain fragments bound to this heavy chain region. Within this heavy chain region are four highly conserved contiguous heptad repeats (775-802) which are predicted to form a tight α- helical coiled-coil interaction with the heptad repeat-containing N-terminus of the light chain, in particular region 106-152 of human light chain. This predicted hydrophobic, α-helical coiled-coil interaction is supported by both circular dichroism spectroscopy of the recombinant kinesin heavy chain fragment 771-963, which displays an α-helical content of 70%, and the resistance of the heavy/light chain interdiction to high salt (0.5 M).