Fluorescence hybridization to interphase nuclei in liquid suspension allows quantification of chromosome-specific DNA sequences using flow cytometry and the analysis of the three-dimensional positions of these sequences in the nucleus using fluorescence microscopy. The three-dimensional structure of nuclei is substantially intact after fluorescence hybridization in suspension, permitting the study of nuclear organization by optical sectioning. Images of the distribution of probe and total DNA fluorescence within a nucleus are collected at several focal planes by quantitative fluorescence microscopy and image processing. These images can be used to reconstruct the three-dimensional organization of the target sequences in the nucleus. We demonstrate here the simultaneous localization of two human chromosomes in an interphase nucleus using two probe labeling schemes (AAF and biotin). Alternatively, dual-beam flow cytometry is used to quantify the amount of bound probe and total DNA content. We demonstrate that the intensity of probe-linked fluorescence following hybridization is proportional to the amount of target DNA over a 100-fold range in target content. This was shown using four human/hamster somatic cell hybrids carrying different numbers of human chromosomes and diploid and tetraploid human cell lines hybridized with human genomic DNA. We also show that populations of male, female, and XYY nuclei can be discriminated by measuring their fluorescence intensity following hybridization with a Y-chromosome-specific repetitive probe. The delay in the increase in Y-specific fluorescence until the end of S-phase in consistent with the results recorded in previous studies indicating that these sequences are among the last to replicate in the genome. A chromosome-17-specific repetitive probe is used to demonstrate that target sequences as small as one megabase (Mb) can be detected using fluorescence hybridization and flow cytometry.(ABSTRACT TRUNCATED AT 250 WORDS)