Increased frequencies of structural and numerical chromosomal aberrations have been observed in the lymphocytes of benzene-exposed workers. Similar aberrations occurring in bone-marrow cells may contribute to the increased incidence of leukemia seen in these populations. Fluorescence in situ hybridization with chromosome-specific DNA probes is a relatively new technique which shows promise for the identification of aneuploidy-inducing agents. In these studies, fluorescence in situ hybridization with several chromosome-specific DNA probes was used to investigate the ability of the benzene metabolite hydroquinone to induce hyperdiploidy in interphase human lymphocytes. Using a classical satellite probe specific for human chromosome 9, a significant dose-related increase in the frequency of cells containing 3 or more hybridization regions was observed following the in vitro exposure of lymphocytes to hydroquinone at concentrations from 75 to 150 microM. At the 100-microM concentration of hydroquinone, the frequency of nuclei containing 3 or more hybridization regions was determined using probes for chromosomes 1, 7 and 9. Significantly higher frequencies of affected nuclei were observed using the chromosome 1 and 9 probes when compared to the chromosome 7 probe. To establish whether this difference was due to the nonrandom involvement of these chromosomes in hydroquinone-induced hyperdiploidy or to chromosomal breakage within the chromosomal region targeted by these probes, a multicolor fluorescence in situ hybridization approach was developed using probes to two adjacent regions on chromosome 1. Using this tandem-labeling approach, the frequency of nuclei with multiple hybridization regions and the origin of the regions was determined by scoring slides labeled simultaneously with the chromosome 7 alpha satellite probe and the adjacent alpha and classical satellite probes for chromosome 1. The results of these studies confirmed that hydroquinone exposure resulted in a significant increase in hyperdiploid nuclei, but indicated that the different frequency of nuclei containing 3 or more hybridization regions observed using the chromosome 1 and 7 probes, was due to breakage within the chromosomal region targeted by the chromosome 1 classical satellite probe. These results indicate that hydroquinone may contribute significantly to the numerical and structural aberrations observed in benzene-exposed workers. In addition, the multicolor fluorescence in situ hybridization approach utilized in these studies promises to be a powerful technique for the detection of chromosomal breakage occurring in interphase human cells.