The 14-3-3 proteins are involved in diverse signal transduction pathways and interact physically with a wide variety of proteins. Here, we report the partial sequence analysis of a human spleen 14-3-3 protein, which was identified as a variant form of the epsilon isoform. A peptide antibody generated to the variant 14-3-3 localizes in the centrosome and spindle apparatus of mouse leukemic FDCP cells by immunofluorescence microscopy. Immunoblots of centrosomes isolated by sucrose density gradient centrifugation of cell lysates disclose only the epsilon and gamma isoforms, while total cellular lysates contain the epsilon, gamma, beta and zeta isoforms of 14-3-3. These data suggest that a subset of total cellular 14-3-3 proteins are localized in the centrosomes and spindle apparatus. A differential localization of the centrosomal 14-3-3 was observed in mouse 3T3 cells. Serum-starved (quiescent) cells lack the centrosomal 14-3-3, but upon serum-stimulation of these quiescent cells, the centrosomal 14-3-3 reappears. We propose that a subset of intracellular 14-3-3 proteins are localized in the centrosome and spindle apparatus, and may in fact, link mitogenic signaling, the cell cycle, and perhaps the centrosome duplication cycle as well.