Multisite phosphorylation of the nuclear lamins is thought to regulate the process of mitotic nuclear envelope breakdown in vivo. Here we investigate the involvement of two proposed human mitotic lamin kinases, beta II protein kinase C (PKC) and p34cdc2/cyclin B kinase, in human lamin B1 phosphorylation in vitro and in intact cells. We find that both kinases can phosphorylate purified soluble lamin B at similar rates. However, beta II PKC phosphorylates interphase nuclear envelope lamin B at more than 200 times the rate of human p34cdc2/cyclin B kinase. beta II PKC-mediated phosphorylation of lamin B is confined to two sites, Ser395 and Ser405, within the carboxyl-terminal domain, whereas human p34cdc2/cyclin B kinase phosphorylates a single site, Ser23, in the amino-terminal domain. A second potential p34cdc2/cyclin B kinase site within the carboxyl-terminal domain, Ser393, is not phosphorylated by human p34cdc2/cyclin B kinase. However, invertebrate p34cdc2/cyclin B kinase from sea star exhibits a different specificity, phosphorylating both amino- and carboxyl-terminal sites. Mitotic human lamin B from intact cells is phosphorylated predominantly in its carboxyl-terminal domain. Comparative tryptic phosphopeptide mapping demonstrates that the beta II PKC site, Ser405, is a prominent target of mitotic lamin B phosphorylation in vivo. beta II PKC translocates to the nucleus during the G2/M phase of cell cycle concomitant with phosphorylation of Ser405, indicating a physiologic role for nuclear beta II PKC activation in mitotic lamin B phosphorylation in vivo. The presence of phosphorylation sites within the carboxyl-terminal domain of mitotic lamin B which are not phosphorylated by either beta II PKC or p34cdc2/cyclin B kinase suggests the involvement of other lamin kinase(s) in G2/M phase lamin B phosphorylation.