Mitotic cyclins are thought to function as key regulatory subunits of the universal M-phase-promoting p34^cdc2 protein kinase. Previous immunolocalization studies have demonstrated that a fraction of p34^cdc2 undergoes cell cycle-dependent accumulation at the centrosome during G₂/M. In order to identify the mitotic cyclins with which this p34^cdc2 fraction interacts, we carefully examined the subcellular distribution of both cyclin A and Bl in HeLa cells. We show here that part of cyclin Bl is associated with duplicating centrosomes throughout its accumulation in the cytoplasm and up to metaphase. In contrast cyclin A does not exhibit centrosomal association except at the onset of mitosis, from preprophase up to metaphase. We also present cytological and biochemical evidence that cyclin Bl is preferentially accumulated as a detergent-insoluble protein independently of the state of microtubule assembly and under experimental conditions where most of p34^cdc2 is soluble. Interestingly, the electrophoretic pattern of the minor insoluble p34^cdc2 fraction was previously shown to be particularly enriched in slow-migrating and presumably hyperphosphorylated isoforms, known to interact specifically with cyclin Bl during interphase. From these results we propose that the interaction of cyclin Bl with the centrosomes and with the cytoplasmic structures is a constitutive feature of the mechanism whereby a fraction of p34^cdc2 is recruited and subsequently targeted to the cyclin B-dependent activation pathway.