The effects of culture time (52, 64 and 76 h) and cytochalasin B (Cyt-B, 3 micrograms/ml) on the frequency of micronuclei (MN) harbouring whole chromosomes and acentric fragments was investigated in purified lymphocyte cultures of five nonsmoking male donors aged 41-50 years. Centromere-positive (C+) MN were identified by fluorescence in situ hybridization, using an alphoid DNA oligomer probe (SO-alpha AllCen) hybridizing to all human centromeres. For each culture time, 2000 cells and 60 MN were scored per donor, both with and without Cyt-B, making a total of 60,000 cells and 1800 MN. The frequency of MN and the proportion of C+ MN were higher at 64 h and 76 h than at 52 h, irrespective of Cyt-B. The culture time-dependent increase in MN frequency was mainly due to C+ MN which were about 1.5-times more frequent at 64 h and 72 h than at 52 h. The frequencies of C+ MN, expressed per 1000 nuclei, were similar with and without Cyt-B, although the prevalence of C+ MN was consistently about 10 percent units higher in the former type of culture. This effect was due to a decreased frequency of centromere-negative (C-) MN in the binucleate cells, possibly reflecting, e.g. increased inclusion of acentric chromosomal fragments within the main nuclei of such cells, enhanced expulsion of C- MN, or selection against binucleate cells carrying such MN. In conclusion, the present findings indicate that MN harbouring whole chromosomes become more frequent at long culture times with and without Cyt-B and that Cyt-B-induced binucleate cells show a reduced frequency of MN containing acentric fragments. Due to the high background of whole-chromosome-containing MN (mean C+ MN proportions ranged from 42.3% to 62.7%), it may be recommended that centromeric fluorescence in situ hybridization is routinely applied when lymphocyte MN are used as a biomarker of human exposure to clastogens.