To study the mechanisms leading to diazepam (DZ)-induced chromosome loss, we evaluated the effect of the drug on the distribution of cytoplasmic and mitotic apparatus proteins using specific antibodies. The use of antibodies directed against dynein and kinetochores (CREST staining) suggested that chromosomes arranged in monopolar spindles were interacting with short fibers originating from the monopole. Interestingly, nearly 50% of DZ-induced monopolar mitoses showed a punctate staining of centrosomes when evaluated with an anti-gamma-tubulin antibody. The extent of phosphorylation of mitotic proteins was not affected by drug treatment, as shown by staining the cells with an antibody against mitotic phosphorylated proteins (MPM-2). After recovery of DZ, nearly 20% of anaphases were abnormal and mainly consisted of multipolar anaphases and lagging chromosomes; this was consistent with a high frequency of kinetochore-containing micronuclei as evaluated by CREST antibody staining in cells that had divided only once after drug removal, i.e. binucleate cells obtained by cytochalasin-B treatment. Our data confirmed that DZ is a powerful inducer of chromosome loss in cultured rodent cells. Moreover, our results indicate that DZ interfered with the correct assembly of centrosomes.