Previously we identified p34cdc2 as one of two protein kinases mediating the hyperphosphorylation and disassembly of vimentin in mitotic BHK-21 cells. In this paper, we identify the second kinase as a 37 kDa protein. This p37 protein kinase phosphorylates vimentin on two adjacent residues (thr-457 and ser-458) which are located in the C-terminal non-alpha-helical domain. Contrary to the p34cdc2 mediated N-terminal phosphorylation (at ser-55) which can disassemble vimentin intermediate filaments (IF) in vitro, p37 protein kinase phosphorylates vimentin-IF without obviously affecting its structure in vitro. We have further examined the in vivo role(s) of vimentin phosphorylation in the disassembly of the IF network in mitotic BHK cells by transient transfection assays. In untransfected BHK cells, the interphase vimentin IF networks are disassembled into non-filamentous aggregates when cells enter mitosis. Transfection of cells with vimentin cDNA lacking the p34cdc2 phosphorylation site (ser55:ala) effectively prevents mitotic cells from disassembling their IF. In contrast, apparently normal disassembly takes place in cells transfected with cDNA containing mutated p37 kinase phosphorylation sites (thr457:ala/ser458:ala). Transfection of cells with vimentin cDNAs lacking both the N- and C-terminal phosphorylation sites yields a phenotype indistinguishable from that obtained with the single N-terminal mutant. Taken together, our results demonstrate that the site-specific phosphorylation of the N-terminal domain, but not the C-terminal domain of vimentin plays an important role in determining the state of IF polymerization and supramolecular organization in mitotic cells.