Lamin B is reversibly methyl-esterified and phosphorylated during the mammalian cell cycle. In order to study the role of methylation in lamin B function, we isolated mitotic cells in the presence of the microtubule inhibitor, nocodazole. Following removal of nocodazole, methylation of mitotic lamin B was found to precede its assembly into the nuclear envelope as cells exited mitosis. Very little additional methylation took place on assembled lamins. We were able to slow the rate of lamin B methylation with methylthioadenosine (MTA). A delay in lamin B methylation was accompanied by a corresponding delay in assembly of lamin B into the envelope. The delay was approximately 20-30 min beyond the typical 60-70 min usually required. Assembly of lamins A and C, which are not methylated, was also delayed by MTA, although to a lesser degree, suggesting that an interaction between the lamins is necessary for formation of the nuclear envelope. Chromatin decondensation was also slowed in the presence of MTA. Other inhibitors of methylation which had no inhibitory effect on the methyl esterification of lamin B were tested and found to have no effect on envelope assembly or chromatin decondensation. These results were obtained with Chinese hamster ovary cells as well as with the stem cell line, PC 13. Dephosphorylation of lamin B normally follows a time course similar to that of nuclear envelope assembly. In the presence of MTA, however, lamin B assembly was slowed with little effect on dephosphorylation. This resulted in a large population of dephosphorylated, but unassembled, lamin B protein, demonstrating that dephosphorylation is not sufficient for envelope assembly. The lack of effect on the time course of dephosphorylation also suggests that MTA is not acting upstream of the methylation event.