As cells enter mitosis, the protein-tyrosine kinase, p60c-src, is known to be extensively phosphorylated on threonine in its amino-terminal region. In the present work, extracts of mitotic cells were searched for the protein kinase responsible for this phosphorylation. HeLa cells and Xenopus eggs were found to contain a mitosis-specific protein kinase activity capable of phosphorylating highly purified p60c-src in vitro on threonine residues. Tryptic phosphopeptide maps indicate that the mitotic HeLa kinase phosphorylates the same sites in vitro as those used during mitosis in vivo. In addition, this mitotic HeLa kinase comigrates on gel filtration with p34cdc2-associated histone H1 kinase, a well known regulator of mitotic events. Finally, antibodies to the C-terminal peptide of human p34cdc2 specifically deplete p60c-src-phosphorylating activity from mitotic extracts. These results suggest that p60c-src may act as an effector of p34cdc2 in certain mitotic processes.