We have adapted the fluorescence in situ hybridization technique to single-cell gel electrophoresis (comet assayed) preparations. Since cells were embedded in agarose, probed regions could be visualized in three dimensions. This system makes it possible to determine the spatial distribution of chromosome-specific DNA sequences at the level of the individual nucleus (nonelectrophoresed) as well as in chromatin fibers of comets (electrostretched chromosomal DNA). This methodology is likely to bring new insights into the field of interphase nuclear ultrastructure. Here, we present the preliminary data obtained with human blood lymphocytes in Gzero after they have been electrophoresed for different times. Chromosome-specific areas (all centromeres, all telomeres, chromosome 7-specific centromere, and long arm of chromosome 3-specific telomere, as well as three segments of the O6-methylguanine-DNA methyltransferase gene) were investigated. Our results are in agreement with the concept that telomeres are in close association with the nuclear membrane and suggest that centromeres are relatively less condensed structures located in the center of the interphase nucleus.